Anti-fugetactic agents for the treatment of ovarian cancer

ABSTRACT

This invention provides methods and compositions for modulating movement of eukaryotic cells with migratory capacity. More specifically, the invention provides anti-fugetactic agents and methods for the use thereof in enhancing an immune response.

RELATED APPLICATIONS/PATENTS & INCORPORATION BY REFERENCE

This application is the U.S. national phase application, pursuant to 35U.S.C. §371, of PCT international application Ser. No.PCT/US2005/040218, filed Nov. 4, 2005, designating the United States andpublished in English on Dec. 28, 2006 as publication WO 2006/137934 A2,which claims priority to U.S. provisional application Ser. No.60/625,733, filed Nov. 5, 2004. The entire contents of theaforementioned patent applications are incorporated herein by thisreference.

STATEMENT OF GOVERNMENTAL INTEREST

This work was funded in part by grant number NHLBI-44851 from theNational Institutes of Health. Accordingly, the United States Governmentmay have certain rights to this invention.

BACKGROUND OF THE INVENTION

Cell movement in response to specific stimuli is observed to occur inprokaryotes and eukaryotes (Doetsch R N and Seymour W F., 1970; Bailey GB et al., 1985). Cell movement seen in these organisms has beenclassified into three types: chemotaxis or the movement of cells along agradient towards an increasing concentration of a chemical; negativechemotaxis which has been defined as the movement down a gradient of achemical stimulus; and chemokinesis or the increased random movement ofcells induced by a chemical agent. The receptors and signal transductionpathways for the actions of specific chemotactically active compoundshave been extensively defined in prokaryotic cells. Study of E. Colichemotaxis has revealed that a chemical which attracts the bacteria atsome concentrations and conditions may also act as a negativechemotactic chemical or chemorepellent at others (Tsang N et al., 1973;Repaske D) and Adler J. 1981; Tisa L S and Adler J., 1995; Taylor B Land Johnson M S., 1998).

Chemotaxis and chemokinesis have been observed to occur in mammaliancells (McCutcheon M W, Wartman W and H M Dixon, 1934; Lotz M and HHarris 1956; Boyden S V 1962) in response to the class of proteins,called chemokines (Ward S G and Westwick J; 1998; Kim C H et al., 1998;Baggiolini M, 1998; Farber J M; 1997). Additionally, Poznansky et al.(U.S. Pat. No. 6,448,054 and WO 2004/053165, which are incorporated byreference in their entirety) have observed chemorepellent, orfugetactic, activity in mammalian cells. Improved control overchemotaxis, chemokinesis and fugetaxis, such as in mammalian systems, isdesirable.

SUMMARY OF THE INVENTION

Agents with migratory-cell repellant activity (hereinafter “fugetacticagents” and “fugetactic activity,” and/or “chemo-fugetaxis”) aredescribed herein. The invention provides pharmaceutical compositionscontaining the foregoing fugetactic agents, and various therapeutic anddiagnostic methods utilizing the foregoing fugetactic agents. Theinvention also provides fugetactic polypeptides and agents which bindsuch polypeptides, including antibodies. The foregoing can be used,inter alia, in the treatment of conditions characterized by a need tomodulate migratory-cell movement in specific sites in a subject.Important such sites include inflammation sites. The invention alsoprovides methods for identifying agents useful in the modulation of suchfugetactic activity.

The present invention is based, in part, upon the appreciation thatdimerization of a G protein coupled receptor and/or receptor ligand isimportant for imparting fugetactic activity. Thus, the invention relatesto fugetactic compounds which comprise ligand dimers, functionalfragments and derivatives thereof and methods of inducing fugetaxis bycontacting such compounds with a cell which expresses the correspondingG protein coupled receptor. The ligand dimers can be made from native orsynthetic ligands of the G protein coupled receptor. For example, theligands can be chemokines. Preferably, the ligand dimers are dimers ofnative ligands. In a particularly preferred embodiment, the fugetacticcompounds are stable in vivo (e.g., resistant to degradation to themonomeric ligand), selectively fugetactic and are substantially inactiveas chemoattractant compounds.

The fugetactic compounds can be any compound resulting in or causingchemokine and/or chemokine receptor dimerization. Chemokines can act asmonomers on chemokine receptors at concentrations below 100 nM, therebyfunctioning as chemoattractants. Certain chemokines, including IL-8 andSDF-1 can also serve as chemorepellents at high concentrations (e.g.,above 100 nM) where much of the chemokine exists as a dimer.Dimerization of the chemokine elicits a differential response in cells,causing dimerization of chemokine receptors, an activity which isinterpreted as a chemorepellent signal. Thus, binding of the dimericchemokine and dimerization of the cognate chemokine receptor on the cellsurface can elicit a differential signal in the cell, including anincreased calcium flux and concomitant alterations of secondarymessenger molecules, to thereby induce fugetaxis.

In another aspect, the invention relates to anti-fugetactic compoundswhich inhibit fugetaxis by inhibiting receptor dimerization. Suchcompounds can include antibodies that target the amino acids in thereceptor necessary for dimerization. In a particularly preferredexample, such anti-fugetactic compounds possess selective antifugetacticactivity, leaving the chemoattractant properties of the receptorsubstantially intact. For example, the anti-fugetactic compound caninterfere with fugetactic ligand activity or receptor binding, therebyallowing the receptor to receive chemoattractant signals.

In one embodiment, the anti-fugetactic compound can be a compound whichinhibits interaction between native ligand dimers and the G proteincoupled receptor. Preferably the inhibition is selective. For example, acompound that competitively inhibits the native ligand dimer can beused. Compounds that competitively inhibit the native ligand dimer maycomprise two protein chains wherein at least one protein chain is aligand derivative which has been modified to delete the receptor bindingfunction. The second protein chain can be a polypeptide that binds thereceptor. Together, the compound is capable of binding to but notactivating the fugetactic activity of the receptor.

Anti-fugetactic agents therefore include any agents that specificallyinhibit chemokine and/or chemokine receptor dimerization, therebyblocking the chemorepellent response to a fugetactic agent. Blocking thechemorepellent effect of high concentrations of a chemokine secreted bya tumor can be accomplished by anti-fugetactic agents which inhibitchemokine dimer formation or chemokine receptor dimer formation. Forexample, antibodies that target and block chemokine receptordimerization, for example, by interfering with the dimerization domainsor ligand binding can be anti-fugetactic agents. Where desired, thiseffect can be achieved without inhibiting the chemotactic action ofmonomeric chemokine.

In other embodiments, the anti-fugetactic agent is a CXCR4 antagonist,CXCR3 antagonist, CXCR4/SDF-1 antagonist or selective PKC inhibitor.Such compounds include AMD3100, KRH-1636, T-20, T-22, T-140, TE-14011,14012 and TN14003; TAK-779, AK602 and SCH-351125; and Tannic acid andNSC 651016 and thalidomide and GF109230X.

In another aspect, the invention further relates to assays foridentifying the compounds described above. Such assays can includecontacting cells (e.g., T-cells) which express the receptor, thereceptor and/or a receptor ligand in the presence of a test compound anddetecting the presence or absence of dimerization of the receptor and/orligand. Preferably, the assay includes the step of detecting thepresence or absence of fugetaxis of cells (e.g., T-cells) underconditions wherein the cells fugetactically respond to the receptor andligand in the absence of such test compound. The assay can optionallycomprise the step of detecting the presence or absence of chemotaxis orchemokinesis of cells (e.g., T-cells) under conditions wherein the cellschemotactically or chemokinetically respond to the receptor and ligandin the absence of such test compound.

In one embodiment, the invention provides a method of identifying afugetactic agent, the method comprising the steps of: contacting anagent suspected of being a fugetactic agent with a cell with migratorycapacity, measuring the zone of clearance relative to the cell; anddetermining that the zone of clearance is of a sufficient size tothereby identify the fugetactic agent. In certain embodiments, the cellwith migratory capacity is a hematopoietic cell, a neural cell, anepithelial cell, a mesenchymal cell, an embryonic stem cell or a germcell.

In another embodiment, the invention includes a method of identifying ananti-fugetactic agent comprising the steps of contacting an agentsuspected of being an anti-fugetactic agent with a polypeptide, such asa polypeptide ligand for a G protein coupled receptor, that willdimerize in the presence of a fugetactic agent, contacting thepolypeptide with a fugetactic agent; and determining that dimerizationof the polypeptide is inhibited, thereby identifying an anti-fugetacticagent.

In yet another embodiment, a method of identifying an anti-fugetacticagent is provided, the method comprising the steps of contacting anagent suspected of being an anti-fugetactic agent with a cell withmigratory capacity in the presence of a fugetactic agent, measuringmovement of the cell with migratory capacity relative to the fugetacticagent, and determining whether the movement of the cell with migratorycapacity is inhibited, thereby identifying an anti-fugetactic agent.

In yet another aspect, the invention further relates to methods ofmodulating fugetactis to provide a therapeutic response in a subject. Inone embodiment, a method of enhancing an immune response in a subjecthaving a condition that involves a specific site, is provided. Themethod involves locally administering to a specific site in a subject inneed of such treatment an anti-fugetactic agent described herein in anamount effective to inhibit immune cell-specific fugetactic activity atthe specific site in the subject. In some embodiments, the specific siteis a site of a pathogenic infection. In certain embodiments, thespecific site is a germ cell-containing site. In further embodiments,the specific site is an area immediately surrounding a tumor. In certainembodiments, the anti-fugetactic agent is a cytokine binding agent. Inother embodiments, the cytokine binding agent is an anti-cytokineantibody or a cytokine agonist. In a preferred embodiment the cytokineis SDF-1α.

In another embodiment, a method of increasing migration of immune cellsto a tumor site is provided, the method comprising administering to anarea immediately surrounding a tumor an anti-fugetactic agent describedherein in an amount effective to inhibit immune cell-specific fugetacticactivity, thereby increasing migration of immune cells to a tumor site.

According to a further aspect of the invention, a method of inhibitingtumor cell metastasis in a subject, is provided. The method involveslocally administering to a tumor site in a subject in need of suchtreatment an anti-fugetactic agent described herein in an amounteffective to inhibit metastasis of tumor cells from the tumor site inthe subject. In certain embodiments, the anti-fugetactic agent is acytokine binding agent. In other embodiments, the cytokine binding agentis an anti-cytokine antibody or a cytokine agonist. In a preferredembodiment the cytokine is SDF-1α.

In yet another embodiment, a method for treating cancer in a subject isprovided, the method comprising inhibiting cell metastasis in thesubject by administering to a tumor site in the subject ananti-fugetactic agent described herein in an amount effective todecrease movement of tumor cells away from the tumor site, therebyinhibiting tumor cell metastasis in the subject and thereby treatingcancer in the subject. The cancer or tumor type can be one presentlyknown to escape immune recognition in the absence of the methodsdescribed herein.

According to a further aspect of the invention, a method ofcontraception in a subject, is provided. The method involvesadministering to a subject in need of such treatment, an anti-fugetacticagent described herein in an amount effective to inhibit germ cellmigration in the subject. In certain embodiments, the anti-fugetacticagent is a cytokine binding agent. In some embodiments, the cytokinebinding agent is an anti-cytokine antibody or a cytokine agonist. In apreferred embodiment the cytokine is SDF-1α.

The invention also relates to kits for using the compounds as describedabove comprising the compounds and instructions for use.

These and other aspects of the invention, as well as various advantagesand utilities, will be more apparent with reference to the detaileddescription of the preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Detailed Description, given by way of example, but notintended to limit the invention to specific embodiments described, maybe understood in conjunction with the accompanying drawings,incorporated herein by reference. Various preferred features andembodiments of the present invention will now be described by way ofnon-limiting example and with reference to the accompanying drawings inwhich:

FIG. 1: SDF-1 expression construct used for transduction of B16/OVAmelanoma cells.

FIG. 2: Bar graphs depicting chemotactic and fugetactic indicescalculated for murine CD8+ T cells subjected to in vitro transmigrationassays in the presence of conditioned medium (wherein B16/OVA.SDF-1 highcells had been incubated), in comparison with rSDF-1 controls.

FIG. 3: In vitro growth (A) and in vivo tumorigenicity (B) ofB16/OVA.pc, B16/OVA.MSCV, B16/OVA.SDF-1 tumor cells.

FIG. 4: Tumor sizes for each mouse at time of sacrifice are shown.

FIG. 5: Paraffin-embedded sections of tumors from immunized mice werestained with H&E (A and D) or with polyclonal α-CD3 Ab (B,C,E, and F).

FIG. 6: Graphic depictions of results of challenge of immunized (withirradiated B16/OVA. SDF-1-high cells) vs. non-immunized mice withB16/OVA.SDF-1-low and -high tumor cells. Results are shown in terms oftumor development/growth and quantitation of T cell infiltrates.

FIG. 7: Early recruitment of CLIO-tat labelled OT-I CD8+ T cells isimpaired in B16/OVA tumors expressing SDF-1.

FIG. 8: Immunohistochemical studies of T-cell infiltration in B16/OVAtumors correlates with MR imaging.

FIG. 9: Bar graph depicting the number of tumor infiltrating CD3+ cellsfound in mice challenged with B16/OVA.SDF-1-low vs. -high cells vs.B16/OVA.MSCV cells.

FIG. 10: Axial Magnetic Resonance (MR) images generated after adoptivetransfer of OT-1 CD8+ T cells incubated with PBS or AMD3100 into micebearing bilateral B16/OVA.MSCV and B16/OVA.SDF-1-high tumors. Imagesdepicting CD3-specific staining of tissues collected after adoptivetransfer and bar graph depicting quantitation of same.

FIG. 11: Potent antitumor activity of persistent adoptively transferredOT-I CD8+ cells is overcome when SDF-1 is locally expressed.

FIG. 12: FACScan identification of adoptively transferred OT-I CTL fromrecovered tumors and lymph nodes (LN).

FIG. 13: Quantitation of the cytotoxicity of OT-I CD8+ T cells againstB16/OVA.MSCV or B16/OVA.SDF-1 target cells was measured in a standard⁵¹Cr-release assay (A) or in a previously described modified assayperformed in flat bottom wells (B).

FIG. 14: Effect of SDF-1 on activation, proliferation and killingefficacy of OT-I T cells.

FIG. 15: FACS plots and graphic analyses depicting CD4, CD8, and CD62Lstaining of thymic cells and recent thymic emigrants (RTE) fromuntreated vs. AMD3100-treated fetal thymus organ cultures (FTOC).

FIG. 16: Mean chymotrypsin index (MCI) calculated for each IL-8 gradientin the presence of GF109203X, 8-Br-cAMP, Rp-cAMPS, or Rp-8-Br-cGMPS.

FIG. 17: Demonstration of T-cell fugetaxis from MSCV.SDF-1 bright cellsproducing high levels of SDF-1 using time-lapse microscopy.

FIG. 18: Fluorescence measurement of calcium flux in neutrophils inresponse to various concentrations of IL-8.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

“Antibodies” as used herein include polyclonal, monoclonal, singlechain, chimeric, humanized and human antibodies, prepared according toconventional methodology.

“Cytokine” is a generic term for nonantibody soluble proteins which arereleased from one cell subpopulation and which act as intercellularmediators, for example, in the generation or regulation of an immuneresponse. See Human Cytokines: Handbook for Basic & Clinical Research(Aggrawal, et al. eds., Blackwell Scientific, Boston, Mass. 1991) (whichis hereby incorporated by reference in its entirety for all purposes).

“CXCR4/SDF-1 antagonist” refers to a compound that antagonizes SDF-1binding to CXCR4.

By “fugetactic activity” it is meant the ability of an agent to repel(or chemorepel) a eukaryotic cell with migratory capacity (i.e., a cellthat can move away from a repellant stimulus). Accordingly, an agentwith fugetactic activity is a “fugetactic agent.” Such activity can bedetected using any of the transmigration systems described herein (seeExamples), or a variety of other systems well known in the art (see,e.g., U.S. Pat. No. 5,514,555, entitled: “Assays and therapeutic methodsbased on lymphocyte chemoattractants,” issued May 7, 1996, to Springer,T A, et al.). A preferred system for use herein is described in U.S.Pat. No. 6,448,054 by Poznansky et al., which is incorporated herein byreference in its entirety.

“Immune cells” as used herein are cells of hematopoietic origin (seelater discussion), that are involved in the specific recognition ofantigens. Immune cells include antigen presenting cells (APCs), such asdendritic cells or macrophages, B cells, T cells, etc. “Mature T cells”as used herein include T cells of a CD4^(lo) CD8^(hi) CD69+ TCR+,CD4^(hi) CD8^(lo) CD69+ TCR+, CD4+ CD3+ RO+ and/or CD8+ CD3+ RO+phenotype. The fugetactic response of the mature T cells to thecompounds of the invention can be measured as described above, oraccording to the transmigration assays described in greater detail inthe U.S. Pat. No. 6,448,054. Other suitable methods will be known to oneof ordinary skill in the art and can be employed using only routineexperimentation.

As used herein, a “subject” is a human, non-human primate, cow, horse,pig, sheep, goat, dog, cat or rodent. In all embodiments, human subjectsare preferred.

II. Compositions and Methods of the Invention

The present invention is based, in part, upon the appreciation thatdimerization of a G protein coupled receptor and/or receptor ligand isimportant for imparting fugetactic activity. Without being bound bytheory, it is believed that these receptor ligands and/or the receptorsdimerize to activate fugetaxis. Thus, the invention relates tofugetactic compounds which comprise ligand dimers, functional fragmentsand derivatives thereof and methods of inducing fugetaxis by contactingsuch compounds with a cell which expresses the corresponding G proteincoupled receptor. The invention further relates to anti-fugetacticcompounds which modulate the fugetactic effect of such G protein coupledreceptors and/or receptor ligands, for example, by inhibiting ligandand/or receptor dimerization.

The invention involves polypeptides of numerous size and type that bindspecifically to chemokine G protein coupled receptors (CXCR-4 and thelike) and the binding partners thereof (chemokines (e.g., SDF-1 andIL-8). Cytokines include, e.g., interleukins IL-1 through IL-15, tumornecrosis factors alpha and beta, interferons alpha, beta, and gamma,tumor growth factor beta (TGF-β), colony stimulating factor (CSF) andgranulocyte monocyte colony stimulating factor (GM-CSF). SDF-1α is acytokine (chemokine) produced by thymic and bone marrow stroma(References 12-15 and U.S. Pat. No. 5,756,084, entitled: “Human stromalderived factor 1α and 1β,” issued May 26, 1998, to Honjo, et al.), thathas been reported as a highly efficacious and highly potent lymphocytechemoattractant at concentrations lower than about 100 ng/ml.

The action of each cytokine on its target cell is mediated throughbinding to a cell surface receptor. Cytokines share many properties ofhormones, but are distinct from classical hormones in that in vivo, theygenerally act locally on neighboring cells within a tissue. Theactivities of cytokines range from promoting cell growth (e.g., IL-2,IL-4, and IL-7), and arresting growth (IL-10, tumor necrosis factor andTGF-β), to inducing viral resistance (IFN α, β, and γ). See FundamentalImmunology (Paul ed., Raven Press, 2nd ed. 1989): Encyclopedia ofImmunology. (Roitt ed., Academic Press 1992) (which are herebyincorporated by reference in their entirety for all purposes). Incertain embodiments, the cytokine is a cytokine with chemoattractantand/or chemokinetic properties. Examples of such cytokines include: PAF,N-formylated peptides, C5a, LTB₄, LXA₄, chemokines: CXC, IL-8, GCP-2,GROα, GROβ, GROγ, ENA-78, NAP-2, IP-10, MIG, I-TAC, SDF-1α, BCA-1, PF4,Bolekine, MIP-1α, MIP-1β, RANTES, HCC-1, MCP-1, MCP-2, MCP-3, MCP-4,MCP-5 (mouse only), Leukotactin-1 (HCC-2, MIP-5), Eotaxin, Eotaxin-2(MPIF2), Eotaxin-3 (TSC), MDC, TARC, SLC (Exodus-2, 6CKine), MIP-3α(LARC, Exodus-1), ELC (MIP-3β), I-309, DC-CK1 (PARC, AMAC-1), TECK,CTAK, MPIF1 (MIP-3), MIP-5 (HCC-2), HCC-4 (NCC-4), MIP-1γ (mouse only),C-10 (mouse only); C: Lymphotactin; CX₃ C: Fracktelkine (Neurotactin).More preferably, the cytokine is a member of the Cys-X-Cys family ofchemokines (chemokines that bind to the CXCR-4 receptor). Preferred suchagents of the invention include SDF-1α, SDF-1β, and met-SDF-1β. Infurther preferred embodiments, such fugetactic agents include otherCXCR-4 receptor ligands. CXCR-4 ligands include, but are not limited to,HIV-1_(IIIB) gp120, small molecules T134, and/or T22([Tyr5,12,Lys7]-polyphemusin II) (Heveker et al., Curr Biol, 1998,8:369-76).

The polypeptides of the invention, including dimers thereof andpolypeptides that modulate dimerization, may be derived from sourcesknown in the art, such as peptide libraries. Such polypeptides can beprovided by degenerate peptide libraries which can be readily preparedin solution, in immobilized form or as phage display libraries.Libraries further can be synthesized of peptides and non-peptidesynthetic moieties.

Standard recombinant techniques can also be used to produce polypeptidesof the invention. The practice of the present invention employs, unlessotherwise indicated, conventional techniques of molecular biology(including recombinant techniques), microbiology, cell biology,biochemistry and immunology, which are within the skill of the art. Suchtechniques are explained fully in the literature, such as, “MolecularCloning: A Laboratory Manual”, second edition (Sambrook, 1989);“Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture”(Freshney, 1987); “Methods in Enzymology”, “Handbook of ExperimentalImmunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells”(Miller and Calos, 1987); “Current Protocols in Molecular Biology”(Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques areapplicable to the production of the polypeptides of the invention, and,as such, may be considered in making and practicing the invention.

According to one aspect of the invention fugetactic compounds describedherein can be used to achieve a therapeutic result in a subject. In oneembodiment, a method of inhibiting migration of immune cells to aspecific site in a subject is provided. The method involves locallyadministering to a specific site in a subject in need of such treatmenta fugetactic agent described herein in an amount effective to inhibitmigration of immune cells to the specific site in a subject.

Thus, the invention provides a method of inhibiting migration of immunecells to a site of inflammation in the subject, “inflammation” as usedherein, is a localized protective response elicited by a foreign(non-self) antigen, and/or by an injury or destruction of tissue(s),which serves to destroy, dilute or sequester the foreign antigen, theinjurious agent, and/or the injured tissue. Inflammation occurs whentissues are injured by viruses, bacteria, trauma, chemicals, heat, cold,or any other harmful stimuli. In such instances, the classic weapons ofthe immune system (T cells, B cells, macrophages) interface with cellsand soluble products that are mediators of inflammatory responses(neutrophils, eosinophils, basophils, kinin and coagulation systems, andcomplement cascade).

A typical inflammatory response is characterized by (i) migration ofleukocytes at the site of antigen (injury) localization; (ii) specificand nonspecific recognition of “foreign” and other (necrotic/injuredtissue) antigens mediated by B and T lymphocytes, macrophages and thealternative complement pathway; (iii) amplification of the inflammatoryresponse with the recruitment of specific and nonspecific effector cellsby complement components, lymphokines and monokines, kinins, arachidonicacid metabolites, and mast cell/basophil products; and (iv) macrophage,neutrophil and lymphocyte participation in antigen destruction withultimate removal of antigen particles (injured tissue) by phagocytosis.The ability of the immune system to discriminate between “self” and“non-self” (foreign) antigens is therefore vital to the functioning ofthe immune system as a specific defense against non-self antigens.

Non-self antigens are those antigens on substances entering a subject,or exist in a subject but are detectably different or foreign from thesubject's own constituents, whereas self antigens are those which, inthe healthy subject, are not detectably different or foreign from itsconstituents.

In another important embodiment, the inflammation is caused by an immuneresponse against self-antigen, and the subject in need of treatmentaccording to the invention has an autoimmune disease. Autoimmune diseaseas used herein, results when a subject's immune system attacks its ownorgans or tissues, producing a clinical condition associated with thedestruction of that tissue, as exemplified by diseases such asrheumatoid arthritis, uveitis. insulin-dependent diabetes mellitus,hemolytic anemias, rheumatic fever, Crohn's disease. Guillain-Banesyndrome, psoriasis, thyroiditis, Graves' disease, myasthenia gravis.glomerulonephritis, autoimmune hepatitis, multiple sclerosis, systemiclupus erythematosus, etc.

Autoimmune disease may be caused by a genetic predisposition alone, bycertain exogenous agents (e.g., viruses, bacteria, chemical agents,etc.), or both. Some forms of autoimmunity arise as the result of traumato an area usually not exposed to lymphocytes, such as neural tissue orthe lens of the eye. When the tissues in these areas become exposed tolymphocytes, their surface proteins can act as antigens and trigger theproduction of antibodies and cellular immune responses which then beginto destroy those tissues. Other autoimmune diseases develop afterexposure of a subject to antigens which are antigenically similar to,that is cross-reactive with, the subject's own tissue. In rheumaticfever, for example, an antigen of the streptococcal bacterium, whichcauses rheumatic fever, is cross-reactive with parts of the human heart.The antibodies cannot differentiate between the bacterial antigens andthe heart muscle antigens, consequently cells with either of thoseantigens can be destroyed.

Other autoimmune diseases, for example, insulin-dependent diabetesmellitus (involving the destruction of the insulin producing beta-cellsof the islets of Langerhans), multiple sclerosis (involving thedestruction of the conducting fibers of the nervous system) andrheumatoid arthritis (involving the destruction of the joint-liningtissue), are characterized as being the result of a mostly cell-mediatedautoimmune response and appear to be due primarily to the action of Tcells (See, Sinha et al., Science, 1990, 248:1380). Yet others, such asmyesthenia gravis and systemic lupus erythematosus, are characterized asbeing the result of primarily a humoral autoimmune response.Nevertheless, inhibition of migration of immune cells to a specific siteof inflammation involved in any of the foregoing conditions according tothe invention, is beneficial to the subject since it inhibits escalationof the inflammatory response, protecting the specific site (e.g.,tissue) involved, from “self-damage.” In preferred embodiments, thesubject has rheumatoid arthritis, multiple sclerosis, or uveitis.

In a further important embodiments, the inflammation is caused by animmune response against non-self-antigens (including antigens ofnecrotic self-material), and the subject in need of treatment accordingto the invention is a transplant recipient, has atherosclerosis, hassuffered a myocardial infarction and/or an ischemic stroke, has anabscess, and/or has myocarditis. This is because after cell (or organ)transplantation, or after myocardial infarction or ischemic stroke,certain antigens from the transplanted cells (organs), or necrotic cellsfrom the heart or the brain, can stimulate the production of immunelymphocytes and/or autoantibodies, which later participate ininflammation/rejection (in the case of a transplant), or attack cardiacor brain target cells causing inflammation and aggravating the condition(Johnson et al., Sem. Nuc. Med. 1989, 19:238: Leinonen et al.,Microbiol. Path., 1990, 9:67; Montalban et al., Stroke, 1991, 22:750).

According to another aspect of the invention, a method of inhibitingendothelial cell migration to a tumor site in a subject, is provided.The method involves locally administering to an area surrounding a tumorsite in a subject in need of such treatment a fugetactic agent describedherein in an amount effective to inhibit endothelial cell migration tothe tumor site in the subject. In certain embodiments, the areasurrounding the tumor site is not immediate to the tumor site. Importantfugetactic agents are as described above.

According to another aspect of the invention, a method of treatinginfertility and premature labor, including premature delivery andimpending miscarriage, is provided. The method involves administering toa subject in need of such treatment a fugetactic agent described hereinin an amount effective to inhibit immune cells from migrating close to agerm cell (including an egg, a sperm, a fertilized egg, or an implantedembryo) in the subject. A germ cell is a cell specialized to producehaploid gametes. It is a cell further differentiated than a stem cell,that can still give rise to more differentiated germ-line cells. Infurther embodiments, the administration is local to a germcell-containing site of the subject. The foregoing methods of therapymay include co-administration of a non-fugetactic agent together with afugetactic agent of the invention that can act cooperatively,additively, or synergistically with the fugetactic agent of theinvention to inhibit migration of immune cells to the site ofinflammation in the subject. According to some embodiments, a fugetacticagent is administered substantially simultaneously with a non-fugetacticagent to inhibit migration of immune cells to a site of inflammation. Bysubstantially simultaneously, it is meant that the fugetactic agent islocally administered to the subject close enough in time with theadministration of the non-fugetactic agent, whereby the non-fugetacticagent may exert a potentiating effect on migration inhibiting activityof the fugetactic agent. Thus, by substantially simultaneously it ismeant that the fugetactic agent is administered before, at the sametime, and/or after the administration of the non-fugetactic agent. Thefugetactic agent can be administered as a polypeptide, and/or a nucleicacid which expresses a fugetactic agent.

In certain embodiments, the non-fugetactic agents areimmunosuppressants. Such immunosuppressants include: Azathioprine;Azathioprine Sodium: Cyclosporine; Daltroban; GusperimusTrihydrochloride; Sirolimus; Tacrolimus.

In other embodiments, the non-fugetactic agents are anti-inflammatoryagents. Such anti-inflammatory agents include: Alclofenac; AlclometasoneDipropionate; Algestone Acetonide; Alpha Amylase; Amcinafal; Amcinafide;Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac;Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen;Benzydamine Hydrochloride; Bromelains; Broperamole; Budesonide;Carprofen; Cicloprofen; Cintazone; Cliprofen; Clobetasol Propionate;Clobetasone Butyrate; Clopirac; Cloticasone Propionate; CormethasoneAcetate; Cortodoxone; Deflazacort; Desonide; Desoximetasone;Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac Sodium;Diflorasone Diacetate; Diflumidone Sodium; Diflunisal; Difluprednate;Diftalone; Dimethyl Sulfoxide; Drocinonide; Endrysone; Enlimomab;Enolicam Sodium; Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole;Fenbufen; Fenclofenac; Fenclorac; Fendosal; Fenpipalone; Fentiazac;Flazalone; Fluazacort; Flufenamic Acid; Flumizole; Flunisolide Acetate;Flunixin; Flunixin Meglumine; Fluocortin Butyl; Fluorometholone Acetate;Fluquazone; Flurbiprofen; Fluretofen; Fluticasone Propionate;Furaprofen; Furobufen; Halcinonide; Halobetasol Propionate; HalopredoneAcetate; Ibufenac; Ibuprofen; Ibuprofen Aluminum; Ibuprofen Piconol;Ilonidap; Indomethacin; Indomethacin Sodium; Indoprofen; Indoxole;Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam; Ketoprofen;Lofemizole Hydrochloride; Lornoxicam; Loteprednol Etabonate;Meclofenamate Sodium; Meclofenamic Acid; Meclorisone Dibutyrate;Mefenamic Acid; Mesalamine; Meseclazone; Methylprednisolone Suleptanate;Morniflumate; Nabumetone; Naproxen; Naproxen Sodium; Naproxol; Nimazone;Olsalazine Sodium; Orgotein; Orpanoxin; Oxaprozin; Oxyphenbutazone;Paranyline Hydrochloride; Pentosan Polysulfate Sodium; PhenbutazoneSodium Glycerate; Pirfenidone; Piroxicam; Piroxicam Cinnamate; PiroxicamOlamine; Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone;Proxazole; Proxazole Citrate; Rimexolone; Romazarit; Salcolex;Salnacedin; Salsalate; Sanguinarium Chloride; Seclazone; Sermetacin;Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate; Talosalate;Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide;Tetrydamine; Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin Sodium;Triclonide; Triflumidate; Zidometacin; Zomepirac Sodium.

According to another aspect, the invention involves a method ofrepelling immune cells from a material surface. “Material surfaces” asused herein, include, but are not limited to, dental and orthopedicprosthetic implants, artificial valves, and organic implantable tissuesuch as a stent, allogeneic and/or xenogeneic tissue, organ and/orvasculature.

Implantable prosthetic devices have been used in the surgical repair orreplacement of internal tissue for many years. Orthopedic implantsinclude a wide variety of devices, each suited to fulfill particularmedical needs. Examples of such devices are hip joint replacementdevices, knee joint replacement devices, shoulder joint replacementdevices, and pins, braces and plates used to set fractured bones. Somecontemporary orthopedic and dental implants, use high performance metalssuch as cobalt-chrome and titanium alloy to achieve high strength. Thesematerials are readily fabricated into the complex shapes typical ofthese devices using mature metal working techniques including castingand machining.

The material surface is coated with an amount of a fugetactic agentdescribed herein effective to repel immune cells. In importantembodiments, the material surface is part of an implant. In importantembodiments, in addition to a fugetactic agent, the material surface mayalso be coated with a cell-growth potentiating agent, an anti-infectiveagent, and/or an antiinflammatory agent. A cell-growth potentiatingagent as used herein is an agent which stimulates growth of a cell andincludes growth factors such as PDGF, EGF, FGF, TGF, NGF, CNTF, andGDNF.

According to another aspect of the invention, a method of identifying afugetactic agent, is provided. The method involves contacting an agentsuspected of being a fugetactic agent with a cell with migratorycapacity, measuring the zone of clearance relative to the cell; anddetermining that the zone of clearance is of a sufficient size tothereby identify the fugetactic agent. In certain embodiments, the cellwith migratory capacity is a hematopoietic cell, a neural cell, anepithelial cell, a mesenchymal cell, an embryonic stem cell or a germcell.

Cells of hematopoietic origin include, but are not limited to,pluripotent stem cells, multipotent progenitor cells and/or progenitorcells committed to specific hematopoietic lineages. The progenitor cellscommitted to specific hematopoietic lineages may be of T cell lineage, Bcell lineage, dendritic cell lineage, Langerhans cell lineage and/orlymphoid tissue-specific macrophage cell lineage. The hematopoieticcells may be derived from a tissue such as bone marrow, peripheral blood(including mobilized peripheral blood), umbilical cord blood, placentalblood, fetal liver, embryonic cells (including embryonic stem cells),aortal-gonadal-mesonephros derived cells, and lymphoid soft tissue.Lymphoid soft tissue includes the thymus, spleen, liver, lymph node,skin, tonsil and Peyer's patches. In other embodiments, the“hematopoietic origin” cells may be derived from in vitro cultures ofany of the foregoing cells, and in particular in vitro cultures ofprogenitor cells.

Cells of neural origin, include neurons and glia, and/or cells of bothcentral and peripheral nervous tissue that express RR/B (sec, U.S. Pat.No. 5,863,744, entitled: “Neural cell protein marker RR/B and DNAencoding same,” issued Jan. 26, 1999, to Avraham, et al.).

Cells of epithelial origin, include cells of a tissue that covers andlines the free surfaces of the body. Such epithelial tissue includescells of the skin and sensory organs, as well as the specialized cellslining the blood vessels, gastrointestinal tract, air passages, ducts ofthe kidneys and endocrine organs.

Cells of mesenchymal origin include cells that express typicalfibroblast markers such as collagen, vimentin and fibronectin.

An embryonic stem cell is a cell that can give rise to cells of alllineages; it also has the capacity to self-renew.

According to yet another aspect of the invention, an anti-fugetacticagent is provided. In one embodiment, the anti-fugetactic agent is anyagent that inhibits chemokine and/or chemokine receptor dimerization.Chemokines act as monomers on chemokine receptors at concentrationsbelow 100 nM, thereby functioning as chemoattractants. Certainchemokines, including IL-8 and SDF-1 can also serve as chemorepellentsat high concentrations (e.g., above 100 nM) where much of the chemokineexists as a dimer. Dimerization of the chemokine elicits a differentialresponse in cells, causing dimerization of chemokine receptors, anactivity which is interpreted as a chemorepellent signal. Thus, bindingof the dimeric chemokine and dimerization of the cognate chemokinereceptor on the cell surface can elicit a differential signal in thecell, including an increased calcium flux and concomitant alterations ofsecondary messenger molecules, to thereby induce fugetaxis.

Anti-fugetactic agents therefore include any agents that specificallyinhibit chemokine and/or chemokine receptor dimerization, therebyblocking the chemorepellent response to a fugetactic agent. Blocking thechemorepellent effect of high concentrations of a chemokine secreted bya tumor can be accomplished by anti-fugetactic agents which inhibitchemokine dimer formation or chemokine receptor dimer formation. Forexample, antibodies that target and block chemokine receptordimerization, for example, by interfering with the dimerization domainsor ligand binding can be anti-fugetactic agents. Where desired, thiseffect can be achieved without inhibiting the chemotactic action ofmonomeric chemokine.

In other embodiments, the anti-fugetactic agent is a CXCR4 antagonist,CXCR3 antagonist, CXCR4/SDF-1 antagonist or selective PKC inhibitor.

Anti-fugetactic agents of the invention can be but are not limited toCXCR4 antagonists, CXCR3 antagonists, CXCR4/SDF-1 antagonists orselective PKC inhibitors.

The CXCR4 antagonist can be but is not limited to AMD3100 (Proc NatlAcad Sci USA. 2003; 100(23):13513-8), KRH-1636 (Proc Natl Acad Sci USA.2003; 100(7):4185-90), T-20 (Org Biomol Chem. 2004; 2(5):660-4), T-22 (JMed. Virol. 2002 October; 68(2):147-55), T-140 (FEBS Lett. 2003;550(1-3):79-83; Curr Drug Targets Infect Disord. 2004; 4(2):103-10),TE-14011 (Biochem Biophys Res Commun. 2004; 320(1):226-32), T-14012(Curr Opin Investig Drugs. 2001; 2(9):1198-202), or TN14003 (FEBS Lett.2004; 569(1-3):99-104; Cancer Res. 2004; 64(12):4302-8) or an antibodythat interferes with the dimerization of CXCR4.

The CXCR3 antagonist can be but is not limited to TAK-779 (J LeukocBiol. 2003; 73(2):273-80), AK602 (J. Virol. 2004; 78(16):8654-62), orSCH-351125 (J. Virol. 2004; 78(8):4134-44; J. Virol. 2003; 77(9):5201-8)or an antibody that interferes with the dimerization of CXCR3.

The CXCR4/SDF-1 antagonist can be but is not limited to Tannic acid(Clin Cancer Res. 2003; 9(8):3115-23), NSC 651016 or (Clin Cancer Res.2002; 8(12):3955-60) or an antibody that interferes with thedimerization of CXCR4 and/or SDF-1.

The selective PKC inhibitor can be but is not limited to thalidomide orGF109230X (J Biol Chem 1991; 266:15771-81).

The invention may also provide an anti-fugetactic compound, such as ananti-cytokine or cytokine receptor antibody. Various kinds ofantibodies, and methods for their production are well known in the air.Thus, anti-cytokine or cytokine receptor antibodies of the invention maycomprise a fragmented or unfragmented antibody that recognizes anepitopic region on a cytokine of interest. As is well-known in the art,only a small portion of an antibody molecule, the paratope, is involvedin the binding of the antibody to its epitope (see, in general, Clark,W. R. (1986) The Experimental Foundations of Modern Immunology Wiley &Sons. Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed.,Blackwell Scientific Publications, Oxford). The pFc′ and Fc regions, forexample, are effectors of the complement cascade but are not involved inantigen binding. An antibody from which the pFc′ region has beenenzymatically cleaved, or which has been produced without the pFc′region, designated an F(ab′)₂ fragment, retains both of the antigenbinding sites of an intact antibody. Similarly, an antibody from whichthe Fc region has been enzymatically cleaved, or which has been producedwithout the Fc region, designated an Fab fragment, retains one of theantigen binding sites of an intact antibody molecule. Proceedingfurther, Fab fragments consist of a covalently bound antibody lightchain and a portion of the antibody heavy chain denoted Fd. The Fdfragments are the major determinant of antibody specificity (a single Fdfragment may be associated with up to ten different light chains withoutaltering antibody specificity) and Fd fragments retain epitope-bindingability in isolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of conspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. Thus, for example, PCT International PublicationNumber WO 92/04381 teaches the production and use of humanized murineRSV antibodies in which at least a portion of the murine FR regions havebeen replaced by FR regions of human origin. Such antibodies, includingfragments of intact antibodies with antigen-binding ability, are oftenreferred to as “chimeric” antibodies.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)₂, Fab, Fv and Fd fragments;chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)₂ fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornon-human sequences.

In yet another aspect, the invention further relates to methods ofmodulating fugetactis to provide a therapeutic response in a subject.Thus, a method of enhancing an immune response in a subject having acondition that involves a specific site, is provided. The methodinvolves locally administering to a specific site in a subject in needof such treatment an anti-fugetactic agent in an amount effective toinhibit immune cell-specific fugetactic activity at a specific site inthe subject. In some embodiments, the specific site is a site of apathogenic infection. Efficient recruitment of immune cells to helpeliminate the infection is therefore beneficial.

Infection can result from exposure to infectious pathogens. Pathogensinclude, for example, viruses, bacteria, parasites, and fungi.

Examples of viruses that have been found in humans include but are notlimited to: Retroviridae (e.g. human immunodeficiency viruses, such asHIV-1 (also referred to as HDTV-III, LAVE or HTLV-III/LAV, or HIV-III;and other isolates, such as HIV-LP; Picornaviridae (e.g. polio viruses,hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses,echoviruses); Calciviridae (e.g. strains that cause gastroenteritis);Togaviridae (e.g. equine encephalitis viruses, rubella viruses);Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow feverviruses); Coronoviridae (e.g. coronaviruses); Rhabdoviridae (e.g.vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g. ebolaviruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus,measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g.influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga viruses,phleboviruses and Nairo viruses); Arena viridae (hemorrhagic feverviruses); Reoviridae (e.g. reoviruses, orbiviruses and rotaviruses);Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvovirida(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesvirus; Poxyviridae (variola viruses, vaccinia viruses, pox viruses); andIridoviridae (e.g. African swine fever virus); and unclassified viruses(e.g. the agent of delta hepatitis (thought to be a defective satelliteof hepatitis B virus), the agents of non-A, non-B hepatitis (class1=internally transmitted; class 2=parenterally transmitted (i.e.Hepatitis C); Norwalk and related viruses, and astroviruses).

Both gram negative and gram positive bacteria serve as antigens invertebrate animals. Such gram positive bacteria include, but are notlimited to, Pasteurella species, Staphylococci species, andStreptococcus species. Gram negative bacteria include, but are notlimited to, Escherichia coli, Pseudomonas species, and Salmonellaspecies. Specific examples of infectious bacteria include but are notlimited to, Helicobacter pyloris, Borelia burgdorferi, Legionellapneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M. avium, M.intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus,Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes,Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae(Group B Streptococcus), Streptococcus (viridans group), Streptococcusfaecalis, Streptococcus bovis, Streptococcus (anaerobic sps.),Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcussp., Haemophilus influenzae, Bacillus antracis, corynebacteriumdiphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae,Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes,Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp.,Fusobacterium nucleatum, Streptobacillus moniliformis, Treponemapallidium, Treponema pertenue, Leptospira, Rickettsia, and Actinomycesisraelli.

Examples of fungi include Cryptococcus neoformans, Histoplasmacapsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydiatrachomatis, Candida albicans.

Other infectious organisms (i.e., protists) include Plasmodium spp. suchas Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, andPlasmodium vivax and Toxoplasma gondii. Blood-borne and/or tissuesparasites include Plasmodium spp., Babesia microti, Babesia divergens,Leishmania tropica, Leishmania spp., Leishmania braziliensis, Leishmaniadonovani, Trypanosoma gambiense and Trypanosoma rhodesiense (Africansleeping sickness), Trypanosoma cruzi (Chagas' disease), and Toxoplasmagondii.

Other medically relevant microorganisms have been described extensivelyin the literature, e.g., see C. G. A Thomas, Medical Microbiology,Bailliere Tindall, Great Britain 1983, the entire contents of which ishereby incorporated by reference.

In certain embodiments, the specific site is a germ cell containingsite. In this case the recruitment of immune cells to these specificsites will help eliminate unwanted germ cells, and/or implanted andnon-implanted embryos. In further embodiments, co-administration ofcontraceptive agents other than anti-fugetactic agents is also provided.Non-anti-fugetactic contraceptive agents are well known in the art.

In further embodiments, the specific site is an area immediatelysurrounding a cancer or tumor. Since most of the known tumors escapeimmune recognition, it is beneficial to enhance the migration of immunecells to the tumor site.

Cancers or tumors include biliary tract cancer; brain cancer, includingglioblastomas and medulloblastomas; breast cancer; cervical cancer;choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer,gastric cancer; hematological neoplasms, including acute lymphocytic andmyelogenous leukemia; multiple myeloma; AIDS associated leukemias andadult T-cell leukemia lymphoma; intraepithelial neoplasms, includingBowen's disease and Paget's disease; liver cancer (hepatocarcinoma);lung cancer; lymphomas, including Hodgkin's disease and lymphocyticlymphomas; neuroblastomas; oral cancer, including squamous cellcarcinoma; ovarian cancer, including those arising from epithelialcells, stromal cells, germ cells and mesenchymal cells; pancreas cancer;prostate cancer; rectal cancer; sarcomas, including leiomyosarcoma,rhabdomyosarcoma, liposarcoma, fibrosarcoma and osteosarcoma; skincancer, including melanoma, Kaposi's sarcoma, basocellular cancer andsquamous cell cancer; testicular cancer, including germinal tumors(seminoma, non-seminoma[teratomas, choriocarcinomas]), stromal tumorsand germ cell tumors; thyroid cancer, including thyroid adenocarcinomaand medullar carcinoma; and renal cancer including adenocarcinoma andWilms tumor. In important embodiments, cancers or tumors escaping immunerecognition include glioma, colon carcinoma, colorectal cancer, lymphoidcell-derived leukemia, choriocarcinoma, and melanoma.

In further embodiments, co-administration of anti-cancer agents otherthan anti-fugetactic agents is also provided. Non-anti-fugetacticanti-cancer agents include: Acivicin; Aclarubicin; AcodazoleHydrochloride; Acronine; Adozelesin; Aldesleukin; Altretamine;Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine;Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa;Azotomycin; Batimastat; Benzodepa; Bicalutamide; BisantreneHydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate;Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone;Caracemide; Carbetimer; Carboplatin; Carmustine; CarubicinHydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin;Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine;Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine;Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel;Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; DroloxifeneCitrate; Dromostanolone Propionate; Duazomycin; Edatrexate; EflorithineHydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine;Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride;Estramustine; Estramustine Phosphate Sodium; Etanidazole; Etoposide;Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine;Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil;Fluorocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; GemcitabineHydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide;Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1;Interferon Alfa-n3; Interferon Beta-I a; Interferon Gamma-I b;Iproplatini; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole;Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium;Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine;Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate;Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium;Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin;Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride;Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran;Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate;Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride;Plicamycin; Plomestane; Podofilox; Porfimer Sodium; Porfiromycin;Prednimustine; Procarbazine Hydrochloride; Puromycin; PuromycinHydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol; SafingolHydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin;Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin;Streptozocin; Sulofenur; Talisomycin; Taxotere; Tecogalan Sodium;Tegafur, Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone;Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin;Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate; TrestoloneAcetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate;Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa;Vapreotide; Verteporlin; Vinblastine Sulfate; Vincristine Sulfate;Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate;Vinleurosine Sulfate; Vinorelbine Tartrate Virlrosidine Sulfate;Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; ZorubicinHydrochloride.

According to a further aspect of the invention, a method of inhibitingtumor cell metastasis in a subject is provided. The method involveslocally administering to a tumor site in a subject in need of suchtreatment an anti-fugetactic agent in an amount effective to inhibitmetastasis of tumor cells from the tumor site in the subject. In certainembodiments, the anti-fugetactic agent is a cytokine dimer. In otherembodiments, the cytokine binding agent is an anti-cytokine antibody ora cytokine agonist. In a preferred embodiment the cytokine is SDF-1α. Infurther embodiments, co-administration of anti-cancer agents other thananti-fugetactic agents is also provided. Anti-cancer and anti-fugetacticagents are as described above.

Tumor cells move through tissues and away from the primary tumor as aresult of chemokines released by adjacent tissues. Tumor cellsmetastasize to specific tissues releasing chemokines which bind thecognate chemokine receptors on the invading tumor cell. Tumor cells (aswell as stromal tissues within the tumor itself) can produce highconcentrations of chemokines that also cause chemorepulsion of tumorcells at the periphery of the tumor, resulting in both local invasionand metastasis. Anti-fugetactic agents of the invention can block thechemorepulsion of tumor cells, thereby reducing or preventing theinvasion and metastasis that cause the spread of cancer.

According to a further aspect of the invention, a method ofcontraception in a subject, is provided. The method involvesadministering to a subject in need of such treatment, an anti-fugetacticagent in an amount effective to inhibit migration of germ cells in thesubject. In certain embodiments, the anti-fugetactic agent is a cytokinebinding agent. In some embodiments, the cytokine binding agent is ananti-cytokine antibody or a cytokine agonist. In a preferred embodimentthe cytokine is SDF-1α. In further embodiments, the administration islocal to a germ cell-containing site of the subject.

The compositions, as described above, are administered in effectiveamounts. The effective amount will depend upon the mode ofadministration, the particular condition being treated and the desiredoutcome. It will also depend upon, as discussed above, the stage of thecondition, the age and physical condition of the subject, the nature ofconcurrent therapy, if any, and like factors well known to the medicalpractitioner. For therapeutic applications, it is that amount sufficientto achieve a medically desirable result. In some cases this is a local(site-specific) reduction of inflammation. In other cases, it isinhibition of tumor growth and/or metastasis.

Generally, doses of active compounds of the present invention would befrom about 0.01 mg/kg per day to 1000 mg/kg per day. It is expected thatdoses ranging from 50-500 mg/kg will be suitable. A variety ofadministration routes are available. The methods of the invention,generally speaking may be practiced using any mode of administrationthat is medically acceptable, meaning any mode that produces effectivelevels of the active compounds without causing clinically unacceptableadverse effects. Such modes of administration include oral, rectal,topical, nasal, interdermal, or parenteral routes. The term “parenteral”includes subcutaneous, intravenous, intramuscular, or infusion.Intravenous or intramuscular routes are not particularly suitable forlong-term therapy and prophylaxis. They could, however, be preferred inemergency situations. Oral administration will be preferred forprophylactic treatment because of the convenience to the patient as wellas the dosing schedule. When peptides are used therapeutically, incertain embodiments a desirable route of administration is by pulmonaryaerosol. Techniques for preparing aerosol delivery systems containingpeptides are well known to those of skill in the art. Generally, suchsystems should utilize components which will not significantly impairthe biological properties of the antibodies, such as the paratopebinding capacity (see, for example, Sciarra and Cutie, “Aerosols,” inRemington's Pharmaceutical Sciences, 18th edition, 1990, pp 1694-1712;incorporated by reference). Those of skill in the art can readilydetermine the various parameters and conditions for producing antibodyor peptide aerosols without resort to undue experimentation.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active agent. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Lower doses will result from other forms ofadministration, such as intravenous administration. In the event that aresponse in a subject is insufficient at the initial doses applied,higher doses (or effectively higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits. Multiple doses per day are contemplated to achieve appropriatesystemic levels of compounds.

The fugetactic agents, fugetactic binding agents, fragments thereof orand/or anti-fugetactic agents may be combined, optionally, with apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid filler, diluents or encapsulating substanceswhich are suitable for administration into a human. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the molecules of the present invention, and witheach other, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficacy.

The invention in other aspects includes pharmaceutical compositions offugetactic agents and anti-fugetactic agents.

When administered, the pharmaceutical preparations of the invention areapplied in pharmaceutically-acceptable amounts and inpharmaceutically-acceptably compositions. Such preparations mayroutinely contain salt, buffering agents, preservatives, compatiblecarriers, and optionally other therapeutic agents. When used inmedicine, the salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically-acceptable salts thereof and are not excludedfrom the scope of the invention. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically-acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts.

Fugetactic and/or anti-fugetactic molecules (nucleic acids orpolypeptides) preferably are produced recombinantly, although suchmolecules may be isolated from biological extracts. Alternatively,direct administration of cells encoding fugetactic and/oranti-fugetactic agents is also contemplated.

Recombinantly produced fugetactic agents such as SDF-1α polypeptides,include chimeric proteins comprising a fusion of a SDF-1α protein withanother polypeptide, e.g., a polypeptide capable of providing orenhancing protein-protein binding, sequence specific nucleic acidbinding (such as GAL4), enhancing stability of the SDF-1α polypeptideunder assay conditions, or providing a detectable moiety, such as greenfluorescent protein. A polypeptide fused to a SDF-1α polypeptide orfragment may also provide means of readily detecting the fusion protein,e.g., by immunological recognition or by fluorescent labeling.

Various techniques may be employed for introducing nucleic acids of theinvention (SDF-1α sense and anti-sense, dominant negative) into cells,depending on whether the nucleic acids are introduced in vitro or invivo in a host. Such techniques include transfection of nucleicacid-CaPO₄ precipitates, transfection of nucleic acids associated withDEAE, transfection with a retrovirus including the nucleic acid ofinterest, liposome mediated transfection, and the like. For certainuses, it is preferred to target the nucleic acid to particular cells. Insuch instances, a vehicle used for delivering a nucleic acid of theinvention into a cell (e.g., a retrovirus, or other virus; a liposome)can have a targeting molecule attached thereto. For example, a moleculesuch as an antibody specific for a surface membrane protein on thetarget cell or a ligand for a receptor on the target cell can be boundto or incorporated within the nucleic acid delivery vehicle. Forexample, where liposomes are employed to deliver the nucleic acids ofthe invention, proteins which bind to a surface membrane proteinassociated with endocytosis may be incorporated into the liposomeformulation for targeting and/or to facilitate uptake. Such proteinsinclude capsid proteins or fragments thereof tropic for a particularcell type, antibodies for proteins which undergo internalization incycling, proteins that target intracellular localization and enhanceintracellular half life, and the like. Polymeric delivery systems alsohave been used successfully to deliver nucleic acids into cells, as isknown by those skilled in the art. Such systems even permit oraldelivery of nucleic acids.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the fugetactic agent, increasing convenience to thesubject and the physician. Many types of release delivery systems areavailable and known to those of ordinary skill in the art. They includepolymer base systems such as poly(lactide-glycolide), copolyoxalates,polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polyanhydrides. Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109.Delivery systems also include non-polymer systems that are: lipidsincluding sterols such as cholesterol, cholesterol esters and fattyacids or neutral fats such as mono- di- and tri-glycerides; hydrogelrelease systems; sylastic systems; peptide based systems; wax coatings;compressed tablets using conventional binders and excipients; partiallyfused implants; and the like. Specific examples include, but are notlimited to: (a) erosional systems in which the anti-inflammatory agentis contained in a form within a matrix such as those described in U.S.Pat. Nos. 4,452,775, 4,667,014, 4,748,034 and 5,239,660 and (b)diffusional systems in which an active component permeates at acontrolled rate from a polymer such as described in U.S. Pat. Nos.3,832,253, and 3,854,480.

A preferred delivery system of the invention is a colloidal dispersionsystem. Colloidal dispersion systems include lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, andliposomes. A preferred colloidal system of the invention is a liposome.Liposomes are artificial membrane vessels which are useful as a deliveryvector in vivo or in vitro. It has been shown that large unilamellarvessels (LUV). which range in size from 0.2-4.0 um can encapsulate largemacromolecules. RNA, DNA, and intact virions can be encapsulated withinthe aqueous interior and be delivered to cells in a biologically activeform (Fraley, et al., Trends Biochem. Sci., (1981) 6:77). In order for aliposome to be an efficient gene transfer vector, one or more of thefollowing characteristics should be present: (1) encapsulation of thegene of interest at high efficiency with retention of biologicalactivity; (2) preferential and substantial binding to a target cell incomparison to non-target cells: (3) delivery of the aqueous contents ofthe vesicle to the target cell cytoplasm at high efficiency: and (4)accurate and effective expression of genetic information.

Liposomes may be targeted to a particular tissue by coupling theliposome to a specific ligand such as a monoclonal antibody, sugar,glycolipid, or protein. Liposomes are commercially available from GibcoBRL, for example, as LIPOFECTIN™ and LIPOFECTACE™, which are formed ofcationic lipids such as N-[1-(2,3dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA) anddimethyl dioctadecylammonium bromide (DDAB). Methods for makingliposomes are well known in the art and have been described in manypublications. Liposomes also have been reviewed by Gregoriadis, G. inTrends in Biotechnology, (1985) 3:235-241.

In one embodiment, the vehicle is a biocompatible microparticle orimplant that is suitable for implantation into the mammalian recipient.Exemplary bioerodible implants that are useful in accordance with thismethod are described in PCT International application No. PCT/US/03307(Publication No. WO 95/24929, entitled “Polymeric Gene DeliverySystem”). PCT/US/03307 describes a biocompatible, preferablybiodegradable polymeric matrix for containing an exogenous gene underthe control of an appropriate promoter. The polymeric matrix is used toachieve sustained release of the exogenous gene in the patient. Inaccordance with the instant invention, the fugetactic and/oranti-fugetactic agents described herein are encapsulated or dispersedwithin the biocompatible, preferably biodegradable polymeric matrixdisclosed in PCT/US/03307.

The polymeric matrix preferably is in the form of a microparticle suchas a microsphere (wherein a fugetactic and/or anti-fugetactic agent isdispersed throughout a solid polymeric matrix) or a microcapsule(wherein a fugetactic and/or anti-fugetactic agent is stored in the coreof a polymeric shell). Other forms of the polymeric matrix forcontaining a fugetactic agent include films, coatings, gels, implants,and stents. The size and composition of the polymeric matrix device isselected to result in favorable release kinetics in the tissue intowhich the matrix is introduced. The size of the polymeric matrix furtheris selected according to the method of delivery which is to be used.Preferably when an aerosol route is used the polymeric matrix andfugetactic agent are encompassed in a surfactant vehicle. The polymericmatrix composition can be selected to have both favorable degradationrates and also to be formed of a material which is bioadhesive, tofurther increase the effectiveness of transfer. The matrix compositionalso can be selected not to degrade, but rather, to release by diffusionover an extended period of time.

In another important embodiment the delivery system is a biocompatiblemicrosphere that is suitable for local, site-specific delivery. Suchmicrospheres are disclosed in Chickering et al., Biotech. And Bioeng.,(1996) 52:96-101 and Mathiowitz et al., Nature, (1997) 386:410-414.

Both non-biodegradable and biodegradable polymeric matrices can be usedto deliver the fugetactic agents of the invention to the subject.Biodegradable matrices are preferred. Such polymers may be natural orsynthetic polymers. Synthetic polymers are preferred. The polymer isselected based on the period of time over which release is desired,generally in the order of a few hours to a year or longer. Typically,release over a period ranging from between a few hours and three totwelve months is particularly desirable. The polymer optionally is inthe form of a hydrogel that can absorb up to about 90% of its weight inwater and further, optionally is cross-linked with multivalent ions orother polymers.

In general, fugetactic and/or anti-fugetactic agents are delivered usinga bioerodible implant by way of diffusion, or more preferably, bydegradation of the polymeric matrix. Exemplary synthetic polymers whichcan be used to form the biodegradable delivery system include:polyamides, polycarbonates, polyalkylenes, polyalkylene glycols,polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols,polyvinyl ethers, polyvinyl esters, poly-vinyl halides,polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes andco-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, celluloseethers, cellulose esters, nitro celluloses, polymers of acrylic andmethacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropylcellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetatebutyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, cellulose sulphate sodium salt, poly(methylmethacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), poly(octadecyl acrylate), polyethylene, polypropylene,poly(ethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinylchloride, polystyrene, polyvinylpyrrolidone, and polymers of lactic acidand glycolic acid, polyanhydrides, poly(ortho)esters, poly(butic acid),poly(valeric acid), and poly(lactide-cocaprolactone), and naturalpolymers such as alginate and other polysaccharides including dextranand cellulose, collagen, chemical derivatives thereof (substitutions,additions of chemical groups, for example, alkyl, alkylene,hydroxylations, oxidations, and other modifications routinely made bythose skilled in the art), albumin and other hydrophilic proteins, zeinand other prolamines and hydrophobic proteins, copolymers and mixturesthereof. In general, these materials degrade either by enzymatichydrolysis or exposure to water in vivo, by surface or bulk erosion.

Examples of non-biodegradable polymers include ethylene vinyl acetate,poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

Bioadhesive polymers of particular interest include bioerodiblehydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell inMacromolecules. (1993) 26:581-587, the teachings of which areincorporated herein, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate).

In addition, important embodiments of the invention include pump-basedhardware delivery systems, some of which are adapted for implantation.Such implantable pumps include controlled-release microchips. Apreferred controlled-release microchip is described in Santini, J T Jr.et al., Nature, 1999, 397:335-338, the contents of which are expresslyincorporated herein by reference.

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions. Long-term release, areused herein, means that the implant is constructed and arranged todelivery therapeutic levels of the active ingredient for at least 30days, and preferably 60 days. Long-term sustained release implants arewell-known to those of ordinary skill in the art and include some of therelease systems described above.

In certain embodiments, the isolated fugetactic agents of the inventionare delivered directly to the site at which there is inflammation, e.g.,the joints in the case of a subject with rheumatoid arthritis, the bloodvessels of an atherosclerotic organ, etc. For example, this can beaccomplished by attaching an isolated fugetactic molecule (nucleic acidor polypeptide) to the surface of a balloon catheter; inserting thecatheter into the subject until the balloon portion is located at thesite of inflammation, e.g. an atherosclerotic vessel, and inflating theballoon to contact the balloon surface with the vessel wall at the siteof the occlusion. In this manner, the compositions can be targetedlocally to particular inflammatory sites to modulate immune cellmigration to these sites. In another example the local administrationinvolves an implantable pump to the site in need of such treatment.

Preferred pumps are as described above. In a further example, when thetreatment of an abscess is involved, the fugetactic agent may bedelivered topically, e.g., in an ointment/dermal formulation.Optionally, the fugetactic molecules of the invention are delivered incombination with a non-fugetactic molecule (e.g., anti-inflammatory,immunosuppressant, etc).

In a preferred embodiment of the invention, the isolated fugetacticagents of the invention are administered to a subject in combinationwith a balloon angioplasty procedure. A balloon angioplasty procedureinvolves inserting a catheter having a deflated balloon into an artery.The deflated balloon is positioned in proximity to the atheroscleroticplaque and the site of inflammation, and is inflated such that theplaque is compressed against the arterial wall. As a result, the layerof endothelial cells on the surface of the artery is disrupted, therebyexposing the underlying vascular smooth muscle cells. The isolatedfugetactic molecule is attached to the balloon angioplasty catheter in amanner which permits release of the isolated fugetactic molecule at thesite of the atherosclerotic plaque and the site of inflammation. Theisolated fugetactic molecule may be attached to the balloon angioplastycatheter in accordance with standard procedures known in the art. Forexample, the isolated fugetactic molecule may be stored in a compartmentof the balloon angioplasty catheter until the balloon is inflated, atwhich point it is released into the local environment. Alternatively,the isolated fugetactic molecule may be impregnated on the balloonsurface, such that it contacts the cells of the arterial wall as theballoon is inflated. The fugetactic molecule also may be delivered in aperforated balloon catheter such as those disclosed in Flugelman, etal., Circulation, v. 85, p, 1110-1117 (1992). See, also, e.g., publishedPCT Patent Application WO 95/23161, for an exemplary procedure forattaching a therapeutic protein to a balloon angioplasty catheter. Thisprocedure can be modified using no more that routine experimentation toattach a therapeutic nucleic acid to the balloon angioplasty catheter.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims. All referencesdisclosed herein are incorporated by reference in their entirety.

The invention will be more fully understood by reference to thefollowing examples. These examples, however, are merely intended toillustrate the embodiments of the invention and are not to be construedto limit the scope of the invention.

EXAMPLES Example I: Anti-fugetactic Agents Decrease Immune Evasion byMelanomas

T-cells are repelled by SDF-1 by a concentration-dependent and CXCR4receptor-mediated mechanism. Repulsion of tumor antigen-specific T-cellsfrom a tumor expressing SDF-1 allows the tumor cells to evade immunecontrol. As shown herein, anti-fugetactic agents restore immune defensesagainst tumors.

I. Materials and Methods

A. C57B1/6 and OT-I mice

C57B1/6 mice between 6 and 10 weeks old were used in all experiments(Jackson Laboratory, Bar Harbor, Me.). The OT-I TCR transgenic mice werekindly provided by W. R. Heath and F. Carbone (Walter and Eliza HallInstitute, Melbourne, Australia). The OT-I TCR is expressed on CD8+ Tcells and is specific for the peptide OVA₂₅₇₋₂₆₄ (SIINFEKL) bound to theclass I MHC molecule H2-Kb (Hogquist, K. A., et al. 1994 Cell 76:17-2).

B. Cell Lines and Preparation of OT-I CTLs

B16 melanoma cells (H2^(b)) stably expressing chicken OVA (B16/OVA.pc)were provided by Drs. E. Lord and J. Frelinger (Brown, D. M., et al.2001 Immunology 102:486-497). OT-I CD8+ T-cells were isolated from thespleens and lymph nodes of OT-I mice using the Magnetic Cell Sorting andSeparation of Biomolecules (MACS) system (Miltenyi Biotec, Auburn,Calif.) and cultured and expanded (Delfs, M. W., et al. 2001Transplantation 71:606-610). For CXCR4 expression, B16 cells and naïveor effector OT-I CD8+ T cells were immunostained using anti-CXCR4-FITCAb (clone 2B11, BD Pharmingen) and analyzed on a FACS (BecktonDickinson) using FlowJo Software (Tree Star, Inc. Ashland, Oreg.).Activated OT-I CD8+ T-cells were also exposed to murine rSDF-1α(PeproTech, Rocky Hill, N.J.) for 24 or 72 hours at final concentrationsof 10, 100 and 1000 ng/ml. Cells were incubated with FITC-conjugatedannexin V and propidium iodide (Detection Kit I, BD Pharmingen).Quantitation of CD69 (clone H1-2F3) and CD25 (clone PC61, all fromBDPharmingen) expression and CFSE staining, was also employed forevaluation of the effect of SDF-1 on OT-1 CD8+ T-cell apoptosis,activation, and proliferation. The percentage of apoptotic cells wasanalyzed by FACS using FlowJo Software.

In order to analyze the effect of SDF-1 on T-cell proliferation, 2×10⁵OT-I total splenocytes or purified CD8+ T-cells were allowed toproliferate for 3 days in 96 round-bottom well plates precoated with 10μg/ml anti-CD3 mAb (BD PharMingen) in the presence of 2 μg/ml solubleanti-CD28 mAb and 50 U/ml murine rIL-2. OT-I T-cells were preincubatedfor 3 hours with murine SDF-1 at final concentrations of 500 or 1000ng/ml. SDF-1 at each concentration was also added every 24 hours toproliferating T-cells.

For CFSE analysis, T-cells were preincubated with 1 μM CFSE (MolecularProbe, Inc., Eugene, Oreg.) in PBS for 10 minutes. At the time ofharvest, cells were washed twice in cold PBS buffer and incubated for 15minutes at 4° C. with anti-CD69 and anti-CD25 mAb, and flow cytometrywas performed on a FACSCalibur (BD Biosciences). Data analysis wasperformed with the FlowJo software (Tree Star, Inc. Ashland, Oreg.). Foreach marker, the threshold of positivity was found beyond thenon-specific binding observed in the presence of irrelevant isotypematched control antibody. Mean log fluorescence intensity (MFI) valueswere obtained by subtracting the MFI of the isotype control from the MFIof the positively stained sample. To evaluate whether the differencesbetween the peaks of cells were statistically significant with respectto controls, the Kolmogorov-Smirnov (K-S) test for analysis ofhistograms was used.

C. Generation of Genetically Modified B16 Cells Expressing SDF-1

The coding region of murine SDF-1α (82-232 bp) was amplified by PCR andcloned into the EGFP encoding bicistronic murine stem cell virus derivedretroviral transfer vector MSCV2.2, using XhoI and EcoRI cloning sites(FIG. 1A). Cloning results were confirmed by DNA sequencing. 293T HEKpackaging cells were co-transfected with the following vectors using thecalcium-phosphate method: EGFP encoding vectors MSCV2.2-SDF-1 orMSCV2.2, a packaging vector pKat, and pCMV-VSV-G (encoding the vesicularstomatitis virus G-glycoprotein). VSV-G pseudotyped retroviral vectorsencoding SDF-1 and GFP or GFP alone were collected at 48 and 72 hourspost-transfection and used for transduction of B16/OVA melanoma cells(Carlesso, N., et al. 1999. Blood 93:838-848). Cell sorting wasperformed using a FACS Vantage cells sorter (Becton Dickenson, FranklinLakes, N.J.) in order to select the brightest 10% of B16/OVA.MSCV andtwo cell populations with bright and low levels of fluorescence termedB16/OVA.SDF-1-high and B16/OVA.SDF-1-low cells (FIG. 1B).

D. Quantitation of SDF-1 Production and Bioactivity

B16/OVA.MSCV and B16/OVA.SDF-1 cells were grown until they were 80-90%confluent. Cells were incubated for a further 24 hours in mediumcontaining 0.5% FCS. Conditioned media (CM) were harvested andconcentrated using Biomax 5K NMWL filter units (Millipore, Bedford,Mass.). Conditioned media were analyzed by Western blotting using mousemonoclonal anti-SDF-1 Ab (R&D Systems, Minneapolis, Minn.), HRP labeledsheep anti-mouse Ab (Amersham Biosciences Corp, Piscataway, N.J.), andECL detection kit (Amersham). The level of SDF-1 in conditioned mediawas also determined by sandwich ELISA (QUANTIKINE, R&D Systems Inc.,Minneapolis, Minn.). Levels of OVA secreted in conditioned media fromB16/OVA.MCSV and B16/OVA.SDF-1 cells cultured for 24 hours were alsomeasured by Elisa (Alpha Diagnostic Int. San Antonio, Tex.).

Quantitative transmigration assays were performed using a transwellsystem as described (Poznansky, M. C., et al. 2000 Nature Med6:543-548). Purified murine CD8+ T cells (6×10⁴ cells) were added to theupper chamber of each well in a total volume of 150 μl Iscove's modifiedmedium. Conditioned media, 20-fold concentrated, undiluted, or diluted1:10 in DMEM containing 0.5% FCS was added in the lower, upper, or bothlower and upper chambers of the transwell to generate a standard“checkerboard” analysis of cell migration. Further control wellsassessed the chemotactic and fugetactic activity of rSDF-1 atconcentrations of 100 ng/ml and 1 μg/ml. Migration of cells wasquantitated as previously described (Poznansky, M. C., et al. 2000Nature Med 6:543-548). Chemotactic index (CI) and fugetactic index (FI)were determined as a ratio between the number of cells migrating inexperimental conditions divided by the number of cells migrating in thecontrol setting with conditioned media in both upper and lower chambers.

E. Tumorigenicity of B16/OVA Cells in Naïve and Immunized Mice

For in vitro growth, 5×10⁵ tumor cells were seeded in T25 flasks (BectonDickinson, Le Pont De Claix, France). Cultures were harvested daily for3 consecutive days and viable cell yield determined. Tumorigenicity wasdetermined by subcutaneous (s.c.) injection of 2×10⁵ viable cells intothe flank of mice in a total volume of 200 μl PBS using a 27 G needle.Tumor growth was also evaluated in naïve mice challenged withB16/OVA.MSCV or B16/OVA.SDF-1-high cells and injected twice a day s.c.with the CXCR4 antagonist, AMD3100 (1.25 mg/kg s.c., Sigma). Tumorgrowth was measured every three or four days using a caliper. Tumorvolume was recorded as the product of two orthogonal diameters (a×b;a=longest diameter; b=orthogonal width) (Hanson, H. L., et al. 2000Immunity 13:265-276). In immunization studies, mice were injected s.c.(at multiple sites) with 1×10⁶ irradiated (8568 cGy) B16/OVA.pc cells ina volume of 200 μl at d 0 and d 10. Fourteen days after immunization,mice were challenged s.c. in both flanks with 2×10⁵ B16/OVA.MSCV orB16/OVA.SDF-1-high cells, and tumor growth was measured as describedabove (Dranoff, G., et al. 1993 Proc Natl Acad Sci USA 90:3539-3543). Insome experiments, mice were immunized with B16/OVA.SDF-1-high cells andthen challenged with B16/OVA.SDF-1-high or B16/OVA.SDF-1-low cells. Micewere sacrificed when one or both tumors reached 200 mm² in volume.

F. Adoptive Immunotherapy, Transfer of Nanoparticle Labeled CTL, andSurvival Analysis

For survival analysis, mice received 1×10⁷ OT-I CD8+ T-cells in 500 μlof HBSS via tail vein injection. Two groups of mice received HBSS ascontrol. 21 d after adoptive transfer of OT-I CTL, each group ofexperimental and control mice was challenged s.c. in the right flankwith 2×10⁵ B16/OVA.SDF-1 cells or B16/OVA.MSCV control cells in 200 μlof HBSS using a 27 G needle (Bathe, O. F., et al. 2001 J Immunol167:4511-4517). Tumor cell growth was recorded every 3-4 days.Detectable tumor was considered to be >5 mm². Mice were sacrificed whentumors reached 400 mm² in size.

Tat peptide derivatized CLIO (CLIO-HD) was synthesized as described(Kircher, M. F., et al. 2003 Cancer Res 63:6838-6846). For eachexperiment, mice were injected s.c. in the right and left flankrespectively with 5×10⁵ B16/OVA.MSCV and B16/OVA.SDF-1-high cells. Whentumors reached 10-15 mm in at least one diameter, OT-I CD8+ T cellsexpanded in vitro (Brown, D. M. et al. 2001 Immunology 102:486-497) wereharvested, incubated with CLIO-HD (300 μg/ml/1×10⁷ cells), and 3×10⁷labeled cells were injected i.p. into mice bearing bilateral tumors ofsimilar size. Distribution of CLIO-HD labeled cells over time wasassessed via MR imaging (Bruker Pharmascan, 4.7 T) at 24 and 48 hoursafter adoptive transfer. T2 images were used to generate 3Dreconstructions, and T2 measurements were used to quantitate tumoralrecruitment of labeled cells as described. (Kircher, M. F. 2003 CancerRes 63:6838-6846).

Mice were anesthetized throughout imaging with 1-2% isoflurane at 1.5liter/min. For the analysis of T cell recruitment, an axial T2-weightedgradient echo sequence (TR=600 ms, TE=6.0, FOV 4.24×2.12 cm, MTX256×128, 4 averages) and an axial T2-weighted fast spin echo sequence(TR=2000 ms, TE=48.6 ms, FOV 4.24×2.12 cm, MTX 256×128, 8 averages) wereused. T2 fit values were derived from a multislice multiecho sequence(TR=2000 ms, TE=6.5×16 ms, FOV 4.24×2.12 cm, MTX 128×128, 2 averages).Regions of interest for the tumors were drawn by hand, and T2 fit valueswere calculated using an in-house program, CMIR-Image (Kircher, M. F.,et al. 2003. Cancer Res 63:6838).

In some experiments, activated OT-1 CD8+ T cells were also preincubatedwith the CXCR4 antagonist, AMD3100, at concentrations of 0.08 and 0.25μg/ml (15 min at RT) in a 12-well plate at a density of 1×10⁷ cells/wellprior to adoptive transfer (Hatse, S., et al. 2002. FEBS Lett 527:255).Survival analysis was evaluated in mice after in vivo generation of atumor-specific memory T cell compartment (Bathe, O. F., et al. 2001. JImmunol 167:4511). 21 days after adoptive transfer of 1×10⁷ OT-1 CD8+ Tcells (in 500 μl of HBSS via tail vein injection), two groups of mice (4or 6 per group) were challenged s.c. in the right flank with 2×10⁵ B16/OVA.SDF-1-high cells or B16/OVA.MSCV control cells in 200 μl of HBSSusing a 27 G needle (Bathe, O. F., et al. 2001. J Immunol 167:4511). Ascontrols, two groups of mice which did not receive adoptive transferwere challenged with tumors. Detectable tumor was considered to be >5mm². Mice were sacrificed when tumors reached 400 mm² in size.

G. Tumor Immunohistochemistry and FACS Analysis of Tumor-InfiltratingLymphocytes.

Immunized mice and mice that received adoptive transfer of OT-I CD8+T-cells were evaluated for T cell infiltration of tumors byimmunohistochemistry. Animals were sacrificed and tumors excised withpreservation of the capsule. Resected tumors were fixed in 10% neutralbuffered formalin and embedded in paraffin. Serial 5 μm sections oftumors were stained with H & E or with polyclonal anti-CD3 Ab or isotypecontrol Ab, followed by incubation with secondary HRP-labeledanti-rabbit Dako EnVision Ab (DakoCytomation). T-cell infiltration wasquantified by counting four random 200× field powers of the tumorsections, each 0.5 mm² in area.

Quantitation of tumor-infiltrating lymphocytes (TIL) by FACS wasperformed in all mice that received OT-I CD8+ T cells for survivalanalysis (Shrikant, P., et al. 1999. Immunity 11:483-493). Mice weresacrificed, and tumors and two draining lymph nodes were harvested. Thetotal number of CD8/TCR-Vβ-5-1/Vα-2-positive cells was calculated fromthe percentage of the total number of cells recovered from 100 mg oftumor. 100 mg of tumor tissue was treated with DMEM medium containingDNAse I 270 IU/ml, collagenase 200 IU/ml, and hyaluronidase 35 IU/ml for1 hour at 37° C., filtered and washed 3 times with HBSS.

After Fc binding by 2.4G2 Ab, 5×10⁵ cells were stained with anti-CD8-APC(clone 53-6.7), Vα-2-TCR-PE (clone B20-1), Vβ-5,1,5,2-TCR-FITC (cloneMR9-4) (BD Pharmingen) Abs and examined on a FACS. Melanin-containingB16 tumor cells were clearly distinguished from TIL by their side andforward scatter by FACS. The total number of OT-I CD8+ cells in eachtumor fragment was calculated from the percentage ofCD8/TCR-Vβ-5-1/Vα-2-positive cells in the total number of cellsrecovered from 100 mg of tumor.

H. Quantitation of CTL Efficacy in Vitro

Cytotoxicity of OT-I CD8+ cells was measured in a standard ⁵¹Cr-releaseassay in round-bottom wells as previously described (Brainard, D. M., etal. 2004. J Virol 78:5184-5193). The assay was also modified andperformed in flat-bottom wells in order to determine the effects of CTLmigration on killing efficacy in the context of target cells expressingSDF-1 (Hahne, M., et al. 1996 Science 274:1363-1366). B16/OVA.MSCV orB16/OVA.SDF-1-high target cells were labeled with 100 μCi of ⁵¹Cr per10⁶ cells for 1 hour at 37° C. After pulsing with 1 μM OVA-specificSIINFEKL-peptide, dilutions of effector cells were added to ⁵¹Cr-labeledtarget cells in 100 μl aliquots to give the indicated effector-to-targetcell ratios (from 30:1 to 1:1) and then incubated at 37° C. in 5% CO₂for 5 hours. 30 μl of supernatant were harvested, and the radioactivitywas counted in a γ-radiation counter. Cytotoxicity was expressed eitheras percent of lysis or as Lytic Unit 30 (LU30) as previously described(Brainard, D. M., et al. 2004. J Virol 78:5184-5193, Bryant, J., et al.1992. J Immunol Meth 146:91-103). ⁵¹Cr release assays were alsoperformed in flat-bottom wells after preincubation of OT-I CD8+ T cellswith the CXCR4 antagonist AMD3100 at concentrations of 0.25 and 1 μg/ml(Hatse, S. 2002 FEBS Lett 527:255-262).

The effect of SDF-1 on CTL efficacy was also measured afterproliferating OT-I CD8+ T cells were cocultured with B16/OVA.SDF-1-highor B16/OVA.MSCV cells. In this assay, 5×10⁶ OT-I CD8+ T cells wereallowed to proliferate for 5 days in 12 well plates in the presence of10 μg/ml anti-CD3, 2 μg/ml anti-CD28 and 50 U/ml IL-2. 5×10⁵B16/OVA.MSCV or B16/OVA. SDF-1-high cells were seeded in a 0.4 μM porepolycarbonate membrane (Corning Life Sciences, Acton, Mass.) and placedin the upper chamber from the first day of culture.

In a parallel experiment, OT-I CD8+ T cells were allowed to proliferatefor 5 days in the presence of rSDF-1 at 500 or 1000 ng/ml concentrationsadded daily to each well. In this set of experiments, CTL killingactivity was measured against B16/OVA.MSCV or B16/OVA. SDF-1-high notpulsed with the SIINFEKL antigen.

I. Analysis of Activation, Proliferation and Apoptosis of OT-1 T-CellsExposed to SDF-1

Activated OT-1 CD8+ T-cells were exposed to murine rSDF-1α (PeproTech,Rocky Hill, N.J.) for 24 or 72 hours at final concentrations of 10, 100and 1000 ng/ml. Cells were incubated with FITC-conjugated annexin V andpropidium iodide (Detection Kit I, BD Pharmingen). The percentage ofapoptotic cells was analyzed by FACS using FlowJo Software (Colamussi,M. L., et al. 2001. J Leukoc Biol 69:263). In order to analyze theeffect of SDF-1 on T-cell proliferation, 2×10⁵ OT-1 total splenocytes orpurified CD8+ T-cells were allowed to proliferate for 3 days in 96round-bottom well plates precoated with 10 μg/ml anti-CD3 mAb (BDPharMingen) in the presence of 2 μg/ml soluble anti-CD28 mAb and 50 U/mlmurine rIL-2. OT-1 T-cells were preincubated for 3 hours with murinerSDF-1 at final concentrations of 500 or 1000 ng/ml. SDF-1 at eachconcentration was also added every 24 hours to proliferating T-cells.

For CFSE analysis, T cells were preincubated with 1 μM CFSE (MolecularProbe, Inc., Eugene, Oreg.) in PBS for 10 minutes. At the time ofharvest, cells were washed twice in cold PBS buffer and incubated for 15minutes at 4° C. with anti-CD69 and anti-CD25 mAb, and flow cytometrywas performed on a FACSCalibur (BD Biosciences). Data analysis wasperformed with the FlowJo software (Tree Star, Inc. Ashland, Oreg.). Foreach marker, the threshold of positivity was found beyond thenon-specific binding observed in the presence of irrelevant isotypematched control antibody. Mean log fluorescence intensity (MFI) valueswere obtained by subtracting the MFI of the isotype control from the MFIof the positively stained sample. The Kolmogorov-Smirnov (K-S) test wasused to evaluate whether the differences between the distributions ofMFI were statistically significant with respect to controls.

J. Modeling of the SDF-1 Gradient Generated in the TumorMicroenvironment

The SDF-1 concentration within and around the periphery of asubcutaneous B16/OVA.SDF-1 tumor was mathematically modeled. The tumorwas modeled as a spherical source of chemokine in an infinitehomogeneous medium of normal tissue. The model proposes that SDF-1 isproduced inside the tumor at the same time as it is dispersed bydiffusion and degraded inside the tumor and the surrounding tissue. Thechemokine concentration c at distance r from the center of the tumor wascalculated at steady state from the mass balance on chemokineconcentration around a radial shell of thickness Δr:

$\begin{matrix}{{V\frac{\partial c}{\partial t}} = \left. \left( {J \times A} \right) \middle| {}_{r}{- \left( {J \times A} \right)} \middle| {}_{r + {\Delta\; r}}{{{+ V} \times G} - {V \times E_{g}}} \right.} & (1)\end{matrix}$

where V is the volume of the tumor, c and J are the concentration andflux of chemokine, D the diffusion coefficient (0.6×10⁻⁷ cm²/s)(Lankelma, J. et al. 2000. Microvasc Res 59:149-161). G the chemokineproduction per unit volume, and E_(g) the rate of degradation of SDF-1.

The chemokine flux J was calculated from the Fick's first law ofdiffusion as:

$\begin{matrix}{J = {{- D}\frac{\partial c}{\partial r}}} & (2)\end{matrix}$

The rate of generation G of SDF-1 was estimated experimentally from themeasured amount of chemokine produced over 24 hours in a cell suspension(58±28.5 ng/10⁶ cells/24 h). The rate of degradation of SDF-1 wasestimated at between 6 and 24 hours and was based on previous reportsfrom in vivo studies (Poznansky, M. C., et al. 2000. Nature Med6:543-548).

Substituting the above in Equation (1) yielded the governing partialdifferential equation for SDF-1 concentration at distance r from thecenter of the tumor:

$\begin{matrix}{\frac{\partial c}{\partial t} = {{D\frac{\partial^{2}c}{\partial t^{2}}} + {\frac{2}{r}\frac{\partial c}{\partial r}} + G - E_{g}}} & (3)\end{matrix}$

Equation (3) was solved using the finite differences method by forwarddifferencing in time and central differencing in space. The equation wassolved in time until to a steady state, where further resolving in timedid not change the concentration distribution of SDF-1.

K. Statistical Analysis

Statistical significance of numerical data was determined using theWilcoxon signed rank exact test or Student's paired t-test. Thestatistical survival analysis was performed using die Mantel-Coxlog-rank test.

II. Results

A. Engineered B16/OVA.SDF-1 Cells Express High or Low Levels ofFunctional SDF-1

Cell sorting was performed to select the transduced B16/OVA.pc cellsthat were the brightest and dimmest B16/OVA.SDF-1 cells, termedB16/OVA.SDF-1-high and B16/OVA.SDF-1-low (FIG. 1B). The cells transducedwith the MSCV vector encoding GFP alone were used as the control cellline in all experiments. Calculation of SDF-1 concentration in theconditioned media from B16/OVA-SDF-1-high and -low cultures as measuredby Western blot and ELISA ranged from 35 to 110 ng/ml (mean=58±28.5 ng)and 2.4 to 13 ng/ml (mean=7.6±4.1), respectively, from 1×10⁶ cellscultured for 24 hours (FIG. 1C). No SDF-1 was detected in conditionedmedia from untransduced B16/OVA cells or B16/OVA cells transduced withMSCV-GFP by Western blotting or ELISA (data not shown). No significantdifference in class I MHC expression and OVA production betweenB16/OVA.SDF-1 transductants and B16/OVA.MSCV cells (Table 1, below) wasdetected. B16/OVA.MSCV and B16/OVA.SDF-1 cells expressed very low levelsof CXCR4 by flow cytometry (Table 1, below).

TABLE 1 Table 1. FACS analysis of MHC class I and CXCR4 expression inB16/OVA.MSCV and B16/OVA.SDF-1 high and low cells. Results are shown asMean Fluorescent Intensity values (MFI). Levels of SDF-1 and OVAsecretion in the supernatant from 1 × 10⁶ B16/OVA.MSCV andB16/OVA.SDF-1-high cells cultured for 24 hours are shown (mean ± SD).OVA SDF-1 MHC-I (pg/ml ± (ng/ml ± CXCR4 (MFI ± SD) SD) SD) (MFI ± SD)B16.OVA/ 16.2 ± 2.1 950 ± 70 — 3.06 ± 0.65 MSCV B16.OVA/ 17.1 ± 1.9 1200± 282   58 ± 28.5 2.45 ± 0.67 SDF-1-high B16.OVA/ 17.9 ± 1.3 994 ± 347.6 ± 4.1 2.88 ± 0.96 SDF-1-low

The effect of the SDF-1/CXCR4 axis on tumor growth was evaluated in miceuntreated and treated with the specific CXCR4 antagonist, AMD3100, invivo from the first day after challenge with tumor cells until theappearance of tumors. The average time to tumor growth to a size of 200mm² size was 22±2 days (average ±SD) and 24±1.3, respectively, forB16/OVA.MSCV treated or untreated with AMD3100 (p=0.32, not shown). Thetime for tumor growth to 200 mm² for AMD3100-untreated and -treatedB16/OVA.SDF-1-high tumors, was 23±1.5 and 23.6±2.08 days, respectively(p=0.72, not shown).

Conditioned media from B16/OVA.SDF-1-high cells and B16/OVA.MCSV cellswere tested for functional activity in transmigration assays (FIG. 2).Undiluted and 1:10 diluted conditioned media from B16/OVA.SDF-1 cellsgenerated a chemotactic index (CI) (ratio between the number of cells inthe experimental setting divided by the number of cells in the controlsetting in which conditioned media was placed in the upper and lowerchamber of the transwell) for primary marine CD8+T-cells of 3.6±0.46(mean±SEM) and 2.7±0.5, respectively (FIG. 2A). Murine rSDF-1 (100ng/ml) gave a CI of 5.9±0.9. Conditioned medium from B16/OVA.SDF-1-highcells was then added to the upper chamber for evaluation of fugetacticactivity (FIG. 2B). The fugetactic index (FI) (ratio between the numberof cells migrating away from conditioned medium in the experimentalsetting divided by the number of cells migrating in the control settingin which conditioned medium was placed in the upper and lower chambersof the transwell) was 6.6±2.6 in the presence of 20-fold concentratedand 0.45±0.025 for 10-fold concentrated conditioned media (FIG. 2B).Murine rSDF-1 (1 μg/ml) added to the upper chamber gave a FI of 7.5±1.1.No chemotactic or fugetactic activity was found when concentrated andundiluted conditioned media from B16/OVA.MSCV were tested in this system(data not shown).

B. Expression of SDF-1 by B16/OVA Cells Does not Affect the Kinetics ofTumor Growth

FACScan analysis of class I MHC expression (anti-H-2K^(−b), cloneAF6-88.5, BD Pharmingen) showed similar levels of expression inB16/OVA.SDF-1 cells (MFI 17.1±1.9 SD) and B16/OVA.MSCV (MFI 16.2±2.1 SD)(p=0.14; data not shown). B16/OVA cells transduced with MSCV or SDF-1expressed very low levels of CXCR4 by flow cytometry (MFI: 3.06±0.65 and2.45±0.67, respectively). The growth of B16/OVA.MSCV cells was notsignificantly different from B16/OVA.SDF-1 cells in vitro (FIG. 3A;p=0.13). B16/OVA.MSCV and B16/OVA.SDF-1 tumors showed similar growthkinetic in vivo, suggesting that SDF-1 had no major effect on tumorgrowth in syngeneic mice in the model described herein (FIG. 3B;p=0.23). In view of the very low level of CXCR4 expression in B16/OVAcells, the effect of the SDF-1/CXCR4 axis on tumour growth was alsoevaluated by in vivo treatment of naïve mice with the CXCR4 antagonist,AMD3100, from the first day after challenge until appearance of tumours.The average time to tumor growth to a size of 200 mm² size was 22±2 days(average ±SD) and 24±1.3, respectively, for B16/OVA.MSCV treated oruntreated with AMD3100 (p=0.32). The time in B16/OVA.SDF-1 tumors was23±1.5 and 23.6±2.08, respectively, for untreated and AMD3100-treatedtumors (p=0.72).

C. Immunized Mice do not Control the Growth of B16OVA-SDF-1-High Tumors

To study the local effect of SDF-1 secretion by a tumor, non-immunizedor immunized mice were challenged with both 2×10⁵ B16/OVA.MSCV andB16/OVA.SDF-1 tumor cells into the right and left flank, respectively(FIG. 4A). Subsequent tumor growth was measured every three or fourdays, and mice were sacrificed when at least one tumor reached 200 mm².When naive non-immunized mice were injected bilaterally withB16/OVA.MSCV and B16/OVA.SDF-1-high cells, tumors in all cases developedto a size of 200 mm², and all mice were sacrificed by d 25 followingchallenge. No mouse died of causes directly or indirectly related to thegrowth of the tumor itself. When comparing the bilateral tumor size ineach mouse, a random pattern of growth was observed (FIG. 4B). In 50% ofanimals, B16/OVA.SDF-1-high tumors grew more rapidly than thecontralateral B16/OVA.MSCV tumor, and there was no significant growthadvantage of SDF-1-expressing tumors over control B16/OVA.MSCV tumors(FIG. 4B; p=0.7). In contrast, immunized mice demonstrated a strikinglydifferent pattern of tumor growth (FIG. 4C). Fifty percent of immunizedmice showed no evidence of B16/OVA.MSCV tumor development in the rightflank at the end of follow-up, whereas all mice developed theB16/OVA.SDF-1-high tumor in the left flank. When comparing the tumorsize in mice that developed bilateral tumors, all mice developed a >200mm² B16/OVA.SDF-1-high tumor when the contralateral B16/OVA.MSCV tumorwas significantly smaller in size (FIG. 4C; p=0.0001).B16/OVA.SDF-1-high tumor growth in immunized mice was significantlydelayed in comparison to tumor growth in non immunized mice(respectively, 29.4±4.9 vs 12.8±2 d, mean±SD, p=0.0002), indicating thatthe immune system plays a role in the control of tumor growth in thismodel.

Immunohistochemistry was performed in order to quantitate T-cellinfiltration into tumors that were not rejected. In non-immunized mice,there was minimal CD3+ T-cell infiltration into both B16/OVA.MSCV andB16/OVA.SDF-1-high tumors (data not shown). In contrast, there wasconsistent CD3+ T-cell infiltration into B16/OVA.MSCV tumors inimmunized mice (FIG. 5, panels A-C). T-cell infiltration intoB16/OVA.SDF-1-high tumors in immunized mice was markedly reduced (FIG.5, panels D-F). The number of CD3+ T-cells (in four 200× fields from 3mice per group) infiltrating B16/OVA.MSCV tumors was 92.2±28.8 cells/mm²(p=0.016), in contrast to 34.5±10.6 cells/mm², mean±SD, infiltratingB16/OVA.SDF-1-high tumors (p=0.001). These data indicate thatintratumoral expression of high concentrations of SDF-1 leads to areduction in TIL.

The effect of vaccination of mice with irradiated B16/OVA cellsexpressing high levels of SDF-1 to protect mice from bilateral challengewith B16/OVA.SDF-1-low and -high cells was also evaluated. As shown inFIG. 6A, naïve mice challenged with bilateral B16/OVA.SDF-1-low and-high cells showed a random pattern of growth (p=0.088). In contrast,vaccination with irradiated B16/OVA cells expressing high levels ofSDF-1 resulted in rejection of 40% of B16/OVA.SDF-1-low tumors, whereasall mice developed B16/OVA.SDF-1-high tumors in the left flank (FIG. 6B;p=0.021). Furthermore, mice that developed bilateral tumors showed aslower rate of growth of B16/OVA.SDF-1-low compared toB16/OVA.SDF-1-high tumors (data not shown). Quantitation of T-cellinfiltration by immunohistochemistry revealed a significantly greaterinfiltration of CD3+ cells into B16/OVA.SDF-1-low tumors (101±10cells/mm²) than into B16/OVA.SDF-1-high tumors (31±5.1 cells/mm²;p=0.007; FIG. 6C). These data support the concept that, although SDF-1does not affect the immunogenicity of B16/OVA cells, it can serve as abidirectional cue for T-cells, attracting at low concentration andrepelling at high concentration.

D. High, But not Low, Levels of SDF-1 Reduce Recruitment of AdoptivelyTransferred OVA-Specific CTL into Tumors

Adoptive transfer of TCR transgenic T-cells into syngeneic recipientsmakes it possible to directly visualize and quantify T-cell responses invivo. It has been shown previously that OT-I CD8+ cells can beefficiently and specifically recruited into tumors as early as 12 hoursafter adoptive transfer into C57BL/6 mice bearing OVA-expressing B16tumors (Kircher, M. F., et al. 2003 Cancer Res 63:6838-6846). In thisstudy, mice were implanted subcutaneously with control B16OVA-MSCV(right flank) and B16OVA-SDF-1-high cells (left flank), so that eachanimal served as its own control. Serial MR images were taken of theanimals when tumors reached 10-15 mm in one diameter. Animals bearingtumors similar in size were imaged at 24 and 48 hours after adoptivetransfer of CLIO-tat labeled OT-1 cells. Lymphocyte infiltration oftumors, as evidenced by quantitation of signal reduction, wasconsistently reduced in B16/OVA.SDF-1-high as compared to B16/OVA.MCSVtumors. In a representative axial slice (FIG. 7), a clear, althoughheterogeneous, signal reduction could be visualized in the B16/OVA.MSCVtumor 24 hours after injection of OT-I CD8+ T-cells, whereas a smallchange in signal intensity was observed in the contralateralB16/OVA.SDF-1-high tumor, indicating that few T-cells were recruited (T2fit value ratio between the right and left tumor: 0.58). MRI at 48 hoursafter CTL injection did not differ significantly from 24-hour analysisfor both B16/OVA.MSCV and B16/OVA.SDF-1-high tumors (data not shown).Tumors were harvested 48 hours after adoptive transfer of labeledT-cells, and sections were stained with H&E and anti-CD3 Ab (FIG. 8).CD3+ T-cell infiltration was clearly observed in B16/OVA.MSCV tumors(FIG. 8A-C), whereas only rare CD3+ T cells were found infiltrating theB16/OVA.SDF-1-high tumor in the same mouse (FIG. 8D-F). Quantitativeanalysis of CD3+ cells in immunohistochemical samples (FIG. 8G) showed a3-fold difference in T-cell infiltration in B16/OVA.MSCV (115.2cells/mm²+35.5, mean±SD), compared to B16/OVA.SDF-1-high tumors (38cells/mm²+17) (p=0.018). No evidence of intravascular accumulation ofT-cells was observed in B16/OVA.SDF-1-high or B16/OVA.MSCV tumors. CD3+T-cells were very rarely seen in control mice that did not receiveadoptively transferred T-cells (data not shown). Thus, early recruitmentof tumor-specific CTL is reduced when SDF-1 is expressed at a high levelin the tumor.

To confirm that SDF-1 impairs the recruitment of OT-I CD8+ T-cells whenexpressed at high but not low levels, the level of T-cell infiltrationwas evaluated in tumors expressing low levels of SDF-1. Mice bearingbilateral B16/OVA.MSCV and B16/OVA.SDF-1-low or B16/OVA.MSCV andB16/OVA.SDF-1-high tumors were adoptively transferred with OT-I CD8+T-cells, and T-cell infiltration was quantitated 48 hours later byimmunohistochemistry. Quantitation of T-cell infiltration showed thatthe expression of low levels of SDF-1 was associated with a highernumber (164±14 cells/mm²) of T-cells infiltrating B16/OVA.SDF-1-lowtumors compared to control B16/OVA.MSCV tumors (101±8.5 cells/mm²)growing in the contralateral thigh (FIG. 9; p=0.032). Therefore,recruitment of adoptively transferred activated tumor antigen-specificCTLs into B16/OVA tumors is significantly increased when SDF-1 isexpressed at a low level and reduced when SDF-1 is expressed at a highlevel by the tumor.

E. Failure of T-Cells to Infiltrate SDF-1 Secreting Tumors isCXCR4-Mediated

In order to investigate whether the expression of high levels of SDF-1did indeed have a direct fugetactic effect on tumor-specific T-cells,the effect was studied of CXCR4 blockade by AMD3100 on the earlyrecruitment of OT-1 T-cells (FIG. 10) (Hatse, S., et al. 2002. FEBS Lett527:255). CLIO-TAT labeled OT-1 CD8+ T-cells were incubated with AMD3100at 0.08 and 0.25 μg/ml and adoptively transferred into mice bearingbilateral tumors. T-cell recruitment was evaluated by MRI and byimmunohistochemistry. A clear MRI signal reduction was observed in theB16/OVA.MSCV tumor and not in the B16/OVA.SDF-1-high tumor 24 hoursafter adoptive transfer of OT-1 T-cells untreated with AMD3100 (FIGS.10A, D). In contrast, T-cell recruitment was clearly visible in theB16/OVA.MSCV, as well as in the B16/OVA.SDF-1-high, tumor when OT-1 CD8+T-cells were pretreated with AMD3100 (FIGS. 10B, E). Quantitation of theT2 fit value in the right and left tumors gave a ratio of 0.55±0.13(mean±SD, n=3) in the AMD3100-pretreated setting, compared to a ratioclose to 1 (I+0.12) when OT-1 T-cells were pretreated with AMD3100before adoptive transfer (p=0.01). To rule out variabilities betweenmice, a second adoptive transfer with OT-1 T-cells pretreated withAMD3100 was performed in the same group of mice that receivedAMD3100-untreated OT-1 T-cells the day before (FIG. 10). Under theseconditions, T-cells again infiltrated both tumors, confirming that CXCR4antagonism by AMD3100 restores T cell recruitment into tumors expressinghigh levels of SDF-1 (FIGS. 10C, F).

Quantitation of T-cell infiltration by immunohistochemistry showed thatincubation of OT-1 CD8+ T-cells with 0.08 μg/ml or 0.25 μg/ml AMD3100before adoptive transfer resulted in a highly significant increase inCD3+ T-cell infiltration into B16/OVA.SDF-1-high tumors (101±10.6 and88.1±16.9 cells/mm²) (p=0.009 and p=0.0111), compared to 40±4.1cells/mm² in AMD3100 untreated B16/OVA.SDF-1-high tumors (FIGS. 10M,N,Oand I,J,O, respectively). In the same groups of mice, the number ofT-cells infiltrating B16/OVA.MSCV tumors (FIGS. 10K, L, and O) was notsignificantly changed (109±3.2 and 93±14.8) compared to the AMD3100untreated B16/OVA.MSCV controls (130±13.9 cells/mm²; p=0.098) (FIGS.10G, H, and O). These data confirm the finding that the primary effectof tumor secretion of SDF-1 is on T-cell infiltration into the tumor-and that a fugetactic effect of the chemokine was abrogated bypretreatment of the tumor antigen-specific T-cells with the CXCR4antagonist, AMD 3100.

F. Secretion of High Levels of SDF-1 by Tumor Cells Impairs Infiltrationand Immune Control by Antigen-Specific Memory T-Cells

Having shown that early recruitment of tumor-specific effector T-cellsis impaired when SDF-1 is present at high levels in the tumormicroenvironment, it was explored whether expression of the chemokinehad an effect on the long-term control of SDF-1-expressing tumors.Therapeutic efficacy of adoptively transferred tumor antigen-specificT-cells is dependent, in part, on the ability of donor cells to persistas long-term memory T-cells (Poznansky, M. C., et al. 2000 Nature Med6:543-548. It has also been shown that the response of T-cells to SDF-1can vary between naïve, effector, and memory CD8+ cells, and that thismay be due to the fact that effector CD8+ T-cells express higher levelsof CXCR4 than memory T-cells (Rempel, S. A., et al. 2000. Clin CancerRes 6:102-111). Flow analysis of OT-I CD8+ cells after 5 d of in vitrostimulation and expansion showed that 67.2%±9.8 (mean±SEM) of effectorcells express CXCR4 compared to 15.5%±1.5 of naïve OT-I cells (data notshown). Whether the recruitment of subpopulations of T-cells to thetumor is dependent on their differential response to SDF-1 was examinedin a model where OT-I CD8+ memory cells are established after adoptivetransfer. OT-I CD8+ cells generated in vitro persist long after adoptivetransfer into syngeneic mice, with the phenotypic and functionalcharacteristics of memory cells. In mice challenged with a thymoma cellline expressing OVA, persistent OT-I CD8+ T-cells showed potentantitumor activity (Bathe, O. F., et al. 2001. J Immunol 167:4511-4517).This model also allowed recovery of antigen-specific CD8+ cells andquantification of the level of T-cell infiltration. Twenty-one daysfollowing adoptive transfer of 1×10⁷ OT-I CD8+ T-cells, two groups ofmice were challenged s.c. with 2×10⁵ B16/OVA.MSCV or B16/OVA.SDF-1-highcells. As in the vaccination protocol, adoptively transferred miceshowed an initial control of B16/OVA.MSCV, as well as ofB16/OVA.SDF-1-high, tumors, as compared to control mice that didn'treceive adoptive transfer (FIG. 11A-D). However, 20% ofadoptively-transferred mice ultimately developed B16/OVA.MSCV tumors by60 days after challenge, as compared to 60% of mice challenged withB16/OVA.SDF-1-high tumors (p=0.036; FIGS. 11B, D and E). IntratumoralOT-I CD8+ T-cells from mice that developed tumors were recovered andunderwent FACS analysis. The total fraction of intratumoral OT-I CD8+T-cells was about 7-fold lower in the B16/OVA.SDF-1-high tumor (FIG.12D) compared to B16/OVA-MSCV tumor (FIG. 12B) (respectively, 0.22%±0.4vs. 1.7%±0.7, mean±SD). When calculating the total number of CD8+T-cells expressing the transgenic Vα2-Vβ5.1/5.2 TCR, a 4.5-foldreduction was found in B16/OVA.SDF-1-high compared to B16/OVA.MSCVtumors (respectively, 12±5.4 vs. 55.5±17.6×10⁴, mean±SD; p=0.005) (FIGS.12B, D, and E). Analysis of OT-I CD8+ T-cells in the draining lymph nodefrom mice bearing B16/OVA.MSCV or B16/OVA.SDF-1-high tumors, as well asfrom mice that rejected tumors, showed no significant difference in thefraction of transgenic CD8+ T-cells (p=0.7; FIG. 12E). These datasupport the view that long-term protection from adoptively transferredOT-I T-cells is overcome when growing tumors express fugetacticconcentrations of SDF-1.

G. SDF-1 Expression at High Levels by Tumor Cells Affects CTL Migrationand Cytotoxicity in Vitro

High expression of SDF-1 by tumor cells protects them from CTL-mediatedlysis in vitro as a result of repulsion of effector cells, mediated viathe chemokine receptor, CXCR4. This was demonstrated by a standard ⁵¹Crrelease assay, using the OT-I CTLs recognizing H-2K^(b) MHC I andOVA₂₅₇₋₂₆₄ peptide, and in a recently developed modified assay, whichtakes account of the influence of effector cell migration oncytotoxicity (Brainard, D. M., et al. 2004. J Virol 78:5184-5193).Ova-specific CTLs killed B16/OVA.MSCV cells as effectively as theykilled B16/OVA-SDF-1-high cells when target cells were effectivelypelleted together in the standard ⁵¹Cr release assay performed inround-bottom wells (FIG. 13A; p=0.16), supporting the view that theproduction of SDF-1 by tumor cells did not influence the susceptibilityof B16/OVA cells in CTL killing. In contrast, when killing activity wasquantitated in flat-bottom wells in which the linear density of cellscould be decreased and, therefore, the distance between effector andtarget cell increased, antigen-specific CTLs were significantly lesseffective at killing B16/OVA cells expressing SDF-1-high as compared toB16/OVA.MSCV cells (FIG. 13B; p=0.0004). To test whether preincubationof OT-I CD8+ T-cells with the CXCR4 antagonist, AMD3100, would restorethe killing activity against B16/OVA cells expressing SDF-1, OT-1 CD8+T-cells were preincubated with the CXCR4 antagonist, AMD3100, at aconcentration of 5 μg/ml. This resulted in a significant increase in thekilling of B16/OVA.SDF-1-high cells (LU30 of untreated OT-I: 49×10⁶±9.2SEM; LU30 of OT-I incubated with AMD3100: 77×10⁶±15 SEM; p=0.045)(FIGS.13C and D). These results were generated in experiments in which tumorcells were pulsed with the SIINFEKL peptide, which allows a higher levelof specific lysis compared to the level of lysis observed from unpulsedtumor cells. Similar results were observed when killing activity wasmeasured against B16/OVA.MSCV or B16/OVA.SDF-1 cells not pulsed with thepeptide (FIG. 14), riling out any difference in OVA expression in the invivo model.

High-level secretion of SDF-1 by tumors cells, therefore, impairs theefficacy of CTL killing in an assay in which CTL migration plays acritical role. Conversely, blockade of the CXCR4 receptor by the highlyspecific CXCR4 antagonist, AMD3100, restores the ability of CTL toengage a tumor cell expressing SDF-1-high. These data are consistentwith the herein-described finding in vivo that blockade of the CXCR4receptor by AMD3100 restores the ability of CTL to infiltrate and engagea tumor cell expressing SDF-1 at a high level.

H. Mathematical Modeling of Chemokine Gradients Predict that ChemokineConcentrations in the Tumor Microenvironment Could Induce T-CellFugetaxis.

The biological activity of SDF-1 depends on its absolute concentrationand the presentation of a gradient of the chemokine within themicroenvironment of the tumor. Precise measurements of localconcentrations of chemokines in tumors are lacking. A model wasestablished for making predictions about SDF-1 concentrations andgradients in the tumor microenvironment based on measured rates ofproduction of SDF-1 by cells in vitro and SDF-1 degradation in vitro(Dunussi-Joannopoulos, K., et al. 2002. Blood 100:1551-1558).

This model predicts that the SDF-1 concentration within 10 to 50 micronsof the periphery of the tumor would be in the range of 0.2 to 0.8 μM fora degradation rate of 6 and 24 hours, respectively. This SDF-1concentration range has been previously shown to induce fugetaxis ofboth resting and activated T-cells in vitro and in vivo (Poznansky, M.C., et al. 2000. Nature Med 6:543-548).

I. SDF-1 Does not Increase Apoptosis or Impair Activation,Proliferation, and Killing Activity of Stimulated OT-I CD8+ T-Cells

Effector OT-I CD8+ cells were incubated for 24 or 72 hours withrecombinant SDF-1 to address the question whether the chemokine had adirect pro-apoptotic effect on T-cells. The percentage of apoptotic OT-ICD8+ cells (PI-negative and annexin V-positive) at 24 hours was 7.9±3.07(mean±SEM) in 10 ng/ml SDF-1, 6.1±3.16 in 100 ng/ml SDF-1, 6.51±2.05 in1 μg/ml SDF-1. These levels of apoptosis did not differ significantlyfrom those seen with OT-I CD8+ cells cultured without SDF-1 (6.67±3.27)(p=0.94, control versus SDF-1 at 1 μg/ml, data not shown). The fractionof apoptotic T-cells was higher after 72 hour incubation in the presenceof 10, 100, or 1000 ng/ml SDF1 (respectively, 19±0.8, 20.3±0.9 and22.9±0.8%), but the level was not significantly different compared tothe fraction of apoptotic cells in the absence of SDF-1 (21.3±1.6,p=0.22, not shown).

Antibody-induced proliferation of OT-I T-cells was measured in thepresence of SDF-1 at chemotactic and fugetactic concentrations (FIG.14A). The level of CD69 and CD25 expression on OT-1 T-cells after threedays of proliferation progressively increased in the presence of SDF-1at concentrations of 500 and 1000 ng/ml (79.4±3.1 vs 89.3±1.74%,respectively, in the absence or presence of SDF-1 at 1000 ng/ml)(p=0.031) (FIG. 14A and Table 2, below). The level of CD25 alsoincreased from 71.9±1.7 in CTL proliferating in the absence of SDF-1 to82.5±1.1 in the presence of 1000 ng/ml SDF-1 (p=0.011). The fraction ofcells that underwent at least one cell division as evaluated by CFSE wasnot significantly different in OT-I CD8+ T-cells proliferating in thepresence of SDF-1 at 500 ng/ml (95.7±1.3%, average ±S.E.M.) and at 1000ng/ml (95.2±2.6%), compared to cells proliferated in absence of SDF-1(95.9±1.9%) (p=0.13) (FIG. 14A and Table 2, below).

TABLE 2 Table 2. Fraction of OT-1 CD8+ T-cells expressing CD25 and CD69and fraction of cells undergoing cell divisions (as measured by CFSE)after activation in the presence of rSDF-1 at different concentrations(A). Fraction of apoptotic OT-1 CD8+ T-cells (PI-negative and annexinV-positive) activated in the presence of rSDF-1 at differentconcentrations (B). Killing activity of OT-I CD8+ T-cells exposed toB16/OVA.MSCV or B16/OVA.SDF-1-high cells. OT-1 T-cells proliferated inthe lower chamber of a transwell system and B16/OVA.MSCV orB16/OVA.SDF-1-high cells were cocultured in the upper chamber (C).Killing activity is expressed as Lytic Unit 30 (LU30) × 10⁶. rSDF-1(ng/ml) (A) 0 500 1000 CD25  71.9 ± 1.77 77.4 ± 0.65 82.5 ± 1.08 CD6979.4 ± 3.1 85.2 ± 1.7  89.3 ± 1.74 CFSE 95.9 ± 1.9 95.7 ± 1.27 95.2 ±2.6  (% dividing cells) rSDF-1 (ng/ml) (B) 0 10 100 1000 % apoptosis21.3 ± 1.69 18.5 ± 0.7 21 ± 0.98 22.9 ± 0.84 Activation in Activation inpresence of presence of (C) B16/OVA.MSCV B16/OVA.SDF-1-high Killingactivity against 32.8 ± 3.64 31.1 ± 3.5 B16/OVA.MSCV Killing activityagainst 30.8 ± 0.9  30.5 ± 3.8 B16/OVA.SDF-1-high

Whether the ability of OT-I CD8+ T-cells to kill B16/OVA cells wasaffected by the presence of SDF-1 (FIG. 14B, Table 2, above) was alsotested. This question was addressed by exposing proliferating OT-I CD8+T-cells during proliferation in vitro to soluble SDF-1 or to SDF-1produced by B16/OVA.SDF-1-high cells growing in the upper chamber of atranswell system. As shown in FIG. 14B, OT-I CD8+ T-cells cocultured inthe presence of SDF-1-high produced by melanoma cells showed the samelevel of killing activity against B16/OVA.MSCV or B16/OVA.SDF-1-hightarget cells compared to the killing activity of T-cells which wereexposed to B16/OVA cells not producing rSDF-1 (respectively, p=0.75 andp=0.31). Similar results were observed when incubating OT-I T-cells withsoluble SDF-1 (data not shown). Therefore, the activation,proliferation, and functional antitumor activity of tumor-specificT-cells was not impaired by high levels of SDF-1.

Local expression of high levels of SDF-1 by engineered tumor cellsabrogates infiltration of the tumor by antigen-specific T-cells. This isthe result of the active movement of T-cells away from the chemokine(i.e., fugetaxis) (Poznansky, M. C. 2000. Nature Med 6:543-548) SDF-1,eotaxin-3, and the HIV-1 envelope protein gp120 have been shown to repelspecific leukocyte subpopulations (Ogilvie, P., et al. 2003. Blood102:789-794, Xiang, Y. Li, et al. 2002. Nature Neurosci 5:843-848, andBrainard, D. M. et al. 2004. J Virol 78:5184-5193). It has beendemonstrated that repulsion of T-cells by high levels of SDF-1 atconcentrations in excess of 100 nM is CXCR4-mediated, pertussistoxin-sensitive, phophatidyl inositol-3 kinase-dependent, and that thesignaling pathway for T-cell fugetaxis is differentially sensitive tointracytoplasmic cAMP concentrations in comparison to SDF-1-inducedT-cell chemotaxis (Poznansky, M. C., et al. 2000. Nature Med 6:543-548).SDF-1 acts as a repellent for T-cells at concentrations that are higherthan the Kd of SDF-1 for CXCR4 in vitro. This occurs in part because ofthe previously reported rapid recycling of the wild type CXCR4 receptorto the cell surface and the postulated existence of a low affinitybinding site for SDF-1 on CXCR4 (Zlatopolskiy, A., et al. 2001. ImmunolCell Biol 79:340-344 and Cyster, J. G. 2002. J Cl Invest 109:1011-1012).

Concentration gradients of SDF-1 predicted to be generated in thissystem in vivo by B16/OVA.SDF-1 cells were mathematically modeled basedon measured levels of production and documented degradation rates forSDF-1 in vivo (Poznansky, M. C., et al. 2000. Nature Med 6:543-548).This model predicts that SDF-1 concentrations would be high enough atthe periphery of the tumor to induce T-cell fugetaxis. The effect onT-cell migration was expected to be predominantly within the localmicroenvironment of the tumor, in view of the fact that SDF-1 hasrecently been shown to be inactivated in the bloodstream and in view ofits high affinity binding to matrix proteins including fibronectin(Villalba, S., et al. 2003. J Leukoc Biol 74:880-888 and Pelletier, A.J., et al. 2000 Blood 96:2682-2690).

SDF-1 regulates the local immune response and is a potentchemoattractant for T cells, pre-B lymphocytes, and dendritic cells(Bleul, C. C., et al. 1996. J Exp Med 184:1101-1109 and D'Apuzzo, M., etal. 1997. Eur J Immunol 27:1788-1793). The expression of SDF-1 by tumorcells would, therefore, be expected to upregulate an immune responseagainst tumor cells. The data presented herein support the view thatSDF-1 may have a bimodal effect on T-cell migration, which includesattraction of T-cells at low concentrations between 10 and 100 nM andrepulsion at concentrations of the chemokine above 100 nM. The immuneresponse to tumor cells, which have been engineered by other groups toexpress SDF-1, has also revealed a dose-dependent effect of thechemokine on this response. Dunussi-Joannopoulos, et al. demonstratedthat low expression of SDF-1 (2 ng/ml) secreted at the tumor site bygenetically modified B16F1 melanoma cells resulted in delayed tumorgrowth and supported the development of long-lived tumor-specific CTLresponses. However, therapeutic immunity against tumors was not observedwhen a high number (>2×10⁵) of irradiated tumor cells expressing highlevels of SDF-1 was used in the vaccination protocol(Dunussi-Joannopoulos, K., et al. 2002. Blood 100:1551-1558). Similarly,immunogenic MethA fibrosarcoma and HM-1 ovarian carcinoma secreting highlevels of SDF-1 (90 ng/mL and 55 ng/mL, respectively) were able toinduce a significant immune response only when tumors were engineered tocoexpress IL-2 or granulocyte-macrophage colony-stimulating factor, aswell as SDF-1 (Nomura, T., et al. 2001. I 91:597-606). Shrikant, et al.recently reported that antigen-specific T-cells adoptively transferredinto mice are able to migrate to the site of tumor challenge where theyproliferate and exert lytic activity only for a short time, but thatthey eventually fail to control tumor growth due to migration away fromthe tumor bulk. Similarly, Hanson, et al. found that tumor-specific CTLadoptively transferred into mice bearing two CMS5 fibrosarcoma tumorsestablished for 3 or 7 d were able to reject early but not late tumorsdue to transient lymphocytic presence at the site of tumors (Cyster, J.G. 2002. J Cl Invest 109:1011-1012). These data are consistent withrecent immunotherapeutic paradigms that predict that early tumors can berejected by the transfer of tumor-specific T-cells, whereas late tumorsare resistant to immune control due to reduced infiltration oftumor-specific T-cells into the tumor itself (Cyster, J. G. 2002. J ClInvest 109:1011-1012). Shown herein is an addition to this model, inwhich growing tumors initially generate SDF-1 at a level which inducesT-cell chemotaxis but ultimately establish an immune privileged sitethrough the activity of high levels of SDF-1 and the repulsion ofeffector T-cells.

Importantly, abrogation of CXCR4 signaling in tumor-specific CTL by thehighly specific CXCR4 antagonist, AMD3100, resulted in restoration oftumor cell lysis, supporting the view that the effects of SDF-1 on CTLrepulsion and killing are CXCR4-mediated.

SDF-1 has been shown to promote the adhesion of lymphocytes toendothelial cells via the induction of the intercellular adhesionmolecule-1 (ICAM-1). However, increased adhesion of T-cells to vesselwalls within tumors expressing high levels of SDF-1 was not evident ineither non-immunized or immunized mice in this study. Results presentedherein support the view that high-level SDF-1 expression in the tumormicroenvironment may result in tight adhesion of a tumor-specific T-cellto the endothelium, but that this is readily followed by SDF-1-mediatedrepulsion from the tumor. Finally, the concomitant challenge of micewith both control and SDF-1-expressing tumors excludes the possibilityof any functional defect of tumor-specific T-cells. Data from combinedMRI and immunohistochemical analysis indicate that local dysregulationof the migration of tumor-specific T-cells results in the failure of theimmune system to control the growth of SDF-1-expressing tumors.

Tumor cells engineered to express high levels of SDF-1 were examined.Primary human melanoma, ovarian and prostate carcinoma, and glioblastomahave been shown to constitutively express high levels of chemokinesincluding SDF-1 and IL-8 (Scotton, C. J., et al. 2002. Cancer Res62:5930-5938, Barbero, S., et al. 2003. Cancer Res 63:1969-1974,Mellado, M., et al. 2001 Curr Biol 11:691-696, and Sun, Y. X., et al.2003 J Cell Biochem 89:462-473). Primary tumor cells including melanomasproduce up to 25.6 ng/ml (per 1×10⁶ cells) of SDF-1 in various systems.These rates of production would generate peak chemokine concentrationsthat are greater than 0.1 μM within and at the periphery of the tumorbased on the mathematical model (Mellado, M., et al. 2001 Curr Biol11:691-696).

Chemokines secreted by primary tumors have been shown to play roles intumorigenesis and the homing of metastasizing tumor cells to specificextratumoral sites in the body. SDF-1 secretion by glioblastoma celllines or ovarian cancer cells has been shown to lead to anautocrine/paracrine regulation of cell growth via the activation of theERK1/2 and Akt pathways and the stimulation of DNA synthesis (Scotton,C. J., et al. 2002. Cancer Res 62:5930-5938 and Barbero, S., et al.2003. Cancer Res 63:1969-1974). Secretion of high levels of SDF-1 by B16OVA tumors did not affect tumor cell growth in vitro and in vivo in thisstudy.

In conclusion, these findings further demonstrate the role of thischemokine as a T-cell fugetactic at a high concentration in a novelmechanism of immune evasion by cancer cells. If repulsion of immunecells from high levels of chemokines elaborated in the microenvironmentaround primary tumors including melanomas contributes to immune evasion,then strategies that overcome this mechanism, including the selectiveantagonism of chemokine/receptor interactions, will be a useful andnovel adjunct to increase the efficacy of cancer vaccines and otheranti-cancer immunotherapeutic approaches.

III. Further Description of the Drawings

FIG. 1: SDF-1 expression construct used for transduction of B16/OVAmelanoma cells (A). GFP expression in sorted B16/OVA.MSCV andB16/OVA.SDF-1 tumor cells by FACScan analysis (B). B16/OVA.pc: (dottedline); B16/OVA.MSCV: (solid line) B16/OVA.SDF-1: (dashed line). Westernblotting analysis of SDF-1 levels secreted by B16/OVA.SDF-1 cells (lane4), compared to rSDF-1 50 ng (lane 1), 20 ng (lane 2), 5 ng (lane 3),and B16/OVA.MSCV (lane 5) (C). M: molecular weight marker.

FIG. 2: B16/OVA.SDF-1-high cells were incubated for 24 hours in DMEMcontaining 0.5% FCS. Conditioned media were then collected and usedundiluted, 1:10 diluted or 20× concentrated in transmigration assays.rSDF-1 at concentrations of 100 ng/ml and 1 μg/ml were used as controls.To evaluate chemotaxis (A) and fugetaxis (B), conditioned media wereplaced in the lower or upper chamber, respectively, and purified murineCD8+ T cells (6×10⁴ cells) were added to the upper chamber of each wellin a total volume of 150 μl. The chemotactic and fugetactic indices werecalculated as the ratio between the number of cells in the experimentalsetting divided by the number of cells in the control setting in whichconditioned media were placed in the upper and lower chambers of thetranswell.

FIG. 3: In vitro growth (A) and in vivo tumorigenicity (B) ofB16/OVA.pc, B16/OVA.MSCV, B16/OVA.SDF-1 tumor cells. *p=0.13; **p=0.23.

FIG. 4: Non-immunized mice were simultaneously inoculated withB16/OVA.MSCV (▪, right flank) and B16/OVA.SDF-1 cells (σ, left flank)(A). Mice (n=8) were sacrificed when at least one tumor reached 200 mm²in size. Tumor sizes for each mouse at time of sacrifice are shown (B).No significant trend in tumor development was observed in naïve mice(B16/OVA.MSCV vs. B16/OVA.SDF-1 tumors) (p=0.7). Tumor growth was alsorecorded in mice that were immunized with irradiated B16/OVA.pc (n=8)prior to inoculation with B16/OVA.MSCV (▪, right flank) andB16/OVA.SDF-1 cells (G, left flank) (C). Tumor growth was absent inimmunized animals B16/OVA.MSCV in 50% of cases and limited the growth of4/8 B16/OVA.MSCV tumors. All (8/8) B16/OVA.SDF-1 tumors implanted in theopposite flank of each immunized mouse rapidly progressed to 200 mm² insize (p=0.0001).

FIG. 5: Paraffin-embedded sections of tumors from immunized mice werestained with H&E (A and D) or with polyclonal α-CD3 Ab (B, C, E, and F).Prominent infiltrates of CD3-positive cells were observed inB16/OVA.MSCV tumors (B and C), but not in tumors expressing SDF-1 (E andF). (Magnification=40× or 200×).

FIG. 6: Mice (groups of 5) were prophylactically immunized withirradiated B16/OVA.SDF-1-high cells prior to inoculation withB16/OVA.SDF-1-low (●, right flank) and B16/OVA.SDF-1-high cells (σ, leftflank) (B). Non-immunized mice were challenged bilaterally with bothtumors as control (A). 40% of mice did not develop the B16/OVA.SDF-1-lowtumors implanted in the right flank (B). All mice showed growth of theB116/OVA.SDF-1-high tumors in the left flank (p=0.021) (B). Nosignificant difference in tumor development was observed in naïve mice(A; p=0.88). α-CD3 staining of tumor sections showed T-cell infiltratesin B16/OVA.SDF-1-low tumors but not in tumors expressing high levels ofSDF-1 (C; *p=0.007).

FIG. 7: Early recruitment of CLIO-tat labeled OT-I CD8+ T-cells isimpaired in B16/OVA tumors expressing SDF-1. Axial MRI slices throughmouse thighs showed a significant signal reduction in the B16/OVA.MSCVtumor compared to the B16/OVA.SDF-1 tumor, indicating that more labeledOT-I T-cells had been recruited into the B16/OVA.MSCV tumor. Theintensity of T-cell recruitment corresponding to dark areas (A) is alsoshown as a T2 spectral color map (B). Number of cells/voxel is indicatedin the color scale. The result shown is representative of fourindependent experiments.

FIG. 8: Immunohistochemical studies of T-cell infiltration in B16/OVAtumors correlates with MR imaging. Tissues were collected 48 hours afteradoptive transfer and sections stained with H&E (A,D) or with polyclonalanti-CD3 Ab (B,C,E,F). Numerous CD3+ T-cells were present within theB16/OVA.MSCV tumor (B,C). In contrast, very few CD3+ T-cells could bedetected in the B16/OVA.SDF-1 tumor (E,F). Results are representative of6 independent experiments. Quantitation of CD3+ cell infiltration intotumors was performed. The mean number (+/−SD) of CD3+ cells per mm² fromsix animals is shown. (G). Magnification=40× or 200×. *: p=0.018.

FIG. 9: Two groups of mice (n=3) were challenged with B16/OVA.MSCV cellsin the right (R) flank and with B16/OVA.SDF-1-low or SDF-1-high cells inthe left (L) flank. After 48 hours from adoptive transfer of OT-1 CD8+T-cells, tumors were collected and sections stained with polyclonalanti-CD3 Ab. A significantly higher number of tumor infiltrating CD3+cells was found in B16/OVA.SDF-1-low compared to B16/OVA.MSCV tumors. Asexpected, mice bearing B16/OVA.MSCV and B16/OVA.SDF-1-high tumors showeda T cell infiltrate in the B16/OVA.MSCV, but not in theB16/OVA.SDF-1-high, tumor. The mean number (+/−SD) of CD3+ cells per mm²from three animals is shown. *: p=0.032; **: p=0.005.

FIG. 10: CLIO-tat-labeled activated OT-1 CD8+ T-cells were incubatedwith PBS (A,D) or AMD3100 (0.08 and 0.25 μg/ml)(B,E and C,F) prior toadoptive transfer into mice bearing bilateral B16/OVA.MSCV andB16/OVA.SDF-1-high tumors. Axial MRI shown in this figure were generated24 hours after adoptive transfer. Panels C and F show MR images of arepresentative mouse (#1), that initially received T-cells untreatedwith AMD3100 and subsequently received a second adoptive transfer withAMD3100-treated OT-1 CD8+ T-cells 24 hours from the first one. MRIimages were obtained again at 24 hours after this second adoptivetransfer. Significant T2 reduction was observed in B16/OVA.MSCV ascompared to B16/OVA.SDF-1-high tumors (D: low T2 fit value ratio).Equivalent T2 reduction was seen when OT-1 T-cells were preincubatedwith AMD3100 with equivalent infiltration of T-cells into B16/OVA.MSCVand B16/OVA.SDF-1-high tumors (E; T2 fit value close to 1). As expected,a second adoptive transfer of AMD3100-pretreated OT-1 CD8+ T-cellsperformed to mouse 1 showed a bilateral T2 signal reduction (F; T2 fitvalue close to 1). Tissues were collected 48 hours after adoptivetransfer and stained with anti-CD3 Ab (G-J; AMD3100 untreated and K-N;AMD3100 treated). The mean number (+/−SD) of CD3+ cells per mm² fromthree animals per group after adoptive transfer of OT-1 T-cellspretreated with AMD3100 at 0.08 and 0.25 ng/ml concentration is shown(O). Magnification=40 and 100×. * p=0.098; ** p=0.011.

FIG. 11: Potent antitumor activity of persistent adoptively transferredOT-I CD8+ cells is overcome when SDF-1 is locally expressed. Groups ofmice (n=6) were challenged with B16/OVA.MSCV (A and B) or B16/OVA.SDF-1(C and D) cells 21 d after intravenous (i.v.) adoptive transfer of 1×10⁷OT-I CD8+ T-cells. Control mice were challenged 21 d after i.v. HBSSalone (A and C). Tumor growth (A-D) and the survival analysis (E) areshown. 35% of B16/OVA.SDF-1-bearing mice (D and E) rejected the tumor,compared to 90% of mice that were challenged with B16/OVA.MSCV tumors (Band E). *: p=0.04.

FIG. 12: FACScan identification of adoptively transferred OT-I CTL fromrecovered tumors and lymph nodes (LN). Each dot plot reports the meanfraction (±SD) of CD8+ cells expressing Vβ5.1/5.2 and Vα2 TCR in thetotal number of B16/OVA.MSCV or B16/OVA.SDF.1 tumor cells analyzed, fromcontrol (A and C) and adoptively transferred mice (B and D). The numberof persistent OT-I CD8+ cells in each tumor and in 2 draining LN wasdetermined (E). AU mice had OT-I CD8+ T-cells in LN without anysignificant difference between groups bearing B16/OVA.MSCV orB16/OVA.SDF-1 tumors (p=0.7). The total number of OT-I CTL inSDF-1-expressing tumors was significantly reduced 4-fold compared tocells recovered from B16/OVA.MSCV tumors (*: p=0.005). Results areexpressed as the mean+/−SD from three independent experiments and threeor four mice per group.

FIG. 13: Quantitation of the cytotoxicity of OT-I CD8+ T-cells againstB16/OVA.MSCV (▪) or B16/OVA.SDF-1 (●) target cells was measured in astandard 51Cr-release assay (A) or in a previously described modifiedassay performed in flat-bottom wells (B). (*: p=0.16, **: p=0.0004).Results of six independent experiments are shown. Cytotoxicity of OT-ICD8+ T-cells pretreated with the specific CXCR4 antagonist, AMD3100 (5μg/ml) (lanes 2 and 4), or untreated (lanes 1 and 3) and incubated withB16/OVA.MSCV (lanes 1 and 2) or B16/OVA.SDF-1 (lanes 3 and 4) was alsoassessed in ⁵¹Cr-release assays in flat-bottom wells (C). Results arereported as LU₃₀/10⁶ OT-I cells, and data are shown as mean±SEM. (*:p=0.045). Results representing mean values+/−SEM from three independentexperiments are shown.

FIG. 14: Effect of SDF-1 on activation, proliferation, and killingefficacy of OT-I T-cells. Splenocytes or purified CD8+ T-cells from OT-Imice were preincubated for 3 hours with SDF-1 at two indicatedconcentrations. Cells were proliferated for three days in 96-well platesin the presence of bound anti-CD3, soluble anti-CD28, and 50 U/ml IL-2,and SDF-1 at the indicated concentration was added to each culture every24 hours (A, upper color dot plots). For analysis of T-cell activation,cells were labeled with anti-CD25 (red histograms), anti-CD69 (A, bluehistograms). Proliferation was measured after 3 days stimulation inCFSE-prelabeled T-cells (A, lower color dot plots). The killing activityof OT-I CD8+ T-cells was evaluated after antibody-induced proliferationof T-cells for 5 days in the presence of B16/OVA-MSCV or B16/OVA.SDF-1cells growing in a 0.4 μM pore polycarbonate membrane placed in theupper chamber from the first day of culture (B). The killing activity ofOT-I CD8+ T-cells stimulated in the presence or absence of SDF-1 wasmeasured against B16/OVA.MSCV or B16/OVA.SDF-1 target cells (not pulsedwith SIINFEKL peptide) in round-bottom well plates after 5 hoursincubation with the chemokine.

Example II: Emigration of CD4 Cells from the Fetal Thymus is Abrogatedby the Anti-fugetactic, CXCR4Antagonist AMD3100

Developing thymocytes undergo maturation whilst migrating through thethymus and, ultimately, emigrate from the thymus to populate peripherallymphoid organs. The process of thymic emigration is controlled in partby the receptor/ligand interaction between SDF-1 and its cognatereceptor CXCR-4. The precise mechanism by which CXCR4/SDF-1 contributesto thymic emigration has now been determined to be regulated by aCXCR4-dependent fugetactic signal that can be abrogated by theanti-fugetactic agent AMD3100.

I. Materials and Methods

A. Mice

E15.5 CXCR4^(−/−) embryos were generated by breeding CXCR4-deficientheterozygous mice on a C57BL/6 background. Presence of the vaginal plugwas considered to represent gestational day 0.5. Mice and embryos weregenotyped as previously described (Ma, Q., et al. 1998. Proc Natl AcadSci USA 95:9448; Ma, Q., et al. 1999. Immunity 10:463). Mice used in thestudy described in the present part were bred in a pathogen-freefacility, in accordance with NIH animal research guidelines.

B. Fetal Thymus Organ Culture

Pregnant females at day 15.5 of gestation were sacrificed by CO₂asphyxiation, and embryos were chilled on ice. Fetal thymuses werecarefully removed under a dissecting microscope, taking care to keep theanatomically identifiable lobes joined together. Thymuses were placed inHBSS and kept on ice. Individual thymuses were gently transferred ontothe surface of a polycarbonate membrane of a 6.5 mm transwell insertwith a 3.0 μm pore size (Corning Incorporated Life Sciences Acton,Mass.). DMEM (360 μl) supplemented with 2 mM glutamine, 100 U/mlpenicillin, 100 μg/ml streptomycin, 2×10⁻⁵ M 2-mercaptoethanol, 0.1 mMnon-essential amino acids, 10% HEPES, and 10% FBS (Sigma) was added tothe lower chamber of the transwell. This amount of medium allowed thethymus to be at the interface of medium and air. The cultures wereincubated at 37° C. in 5% CO, and medium was replaced every 3 days.Thymic emigrants were collected from each thymus by transferring eachtranswell insert containing one thymus onto to a new well 24 hoursbefore harvesting emigrants present in the lower chamber for analysis.On the day of analysis, two thymuses were harvested, and emigrants werepooled from at least 4 thymic lobes from CXCR4^(+/−) and CXCR4^(−/−)mice. For each timepoint, CXCR4^(+/−) and CXCR4^(−/−) thymuses from oneor two pregnant females bred at the same time were analyzed, aiming tominimize differences in the stage of maturation and total cell recovery.E15.5 WT (CXCR4^(+/+)) thymuses were obtained from fetuses from C57BL/6mice breedings. For inhibition studies on thymic emigration, PTX (100ng/ml, Sigma) or murine recombinant SDF-1 (50, 500, 1000 nM, PeproTech,Rocky Hill, N.J.) was added to WT FTOC at day 14 and thymus andemigrants were harvested at day 15 of culture (Poznansky, M. C., et al.2002. J Clin Invest 109:1101). FTOC from DPC 15.5 CXCR4+/− or CXCR4−/−embryos were also prepared as above, and AMD3100 (0.5 or 1 gg/ml; Sigma)was added to the lower chamber of day 14 FTOC. Thymic emigrants werethen collected after 24 hours (Hatse, S., et al. 2002. FEBS Lett527:255).

C. Immunofluorescence Staining and Flow Cytometric Analysis

Thymic lobes were harvested and transferred to a 4-ml round bottompolystyrene tube containing 0.4 mg/ml collagenase (from Clostridiumhistolyticum, type V, clostridiopeptidase A, SIGMA Chemicals), 10% FBS,EDTA 0.2 mg/ml and 0.2 M phosphate. After 30 minutes incubation at 37°C., thymuses were disaggregated by gentle pipetting until no visiblefragments remained. Cell suspensions were washed once in HBSS and 10%FBS to prevent further enzyme action. Cells were resuspended in PBScontaining 0.1% bovine serum albumin and counted using a hemocytometer.After incubation for 30 minutes on ice with 0.5 gg of 2.4G2 antibody toblock Fc binding, direct staining for three or four color analysis wasperformed. Cell suspensions were incubated for 15 minutes at 4° C. andthen washed twice with staining buffer and analyzed using a FACSCalibur(BD Biosciences). Data analysis was performed with FlowJo software (TreeStar, Inc. Ashland, Oreg.). The following monoclonal antibodies wereused: Anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7), anti-CD62L (cloneMEL-14), anti-CD3c (clone 145-2C11), and anti-CXCR4 (clone 2B11/CXCR4),(BD PharMingen, Mountain View, Calif.). For annexin V and PI staining,an Annexin V-FITC Apoptosis Detection Kit I was used (BD Pharmingen).For each marker, the threshold of positivity was found beyond thenon-specific binding observed in the presence of irrelevant controlantibody. Mean log fluorescence intensity (MFI) values were obtained bysubtracting the MFI of the isotype control from the MFI of thepositively stained sample. To evaluate whether the differences betweenthe peaks of cells were statistically significant with respect tocontrols, the Kolmogorov-Smirnov test for analysis of histograms wasused.

D. Transmigration Assays

Quantitative transmigration assays were performed using a transwellsystem (Corning Costar Inc., Corning, N.Y., USA) (6.5-mm diameter, 5-μmpore size, polycarbonate membrane) as described previously (Poznansky,M. C., et al. 2000. Nat Med 6:543). Purified fetal (E.16) thymocytesubpopulations (5×10⁴ cells) (double negative (DN), double positive (DP)and single positive (SP) thymocytes were prepared by FACS sorting aspreviously described (Poznansky, M. C., et al. 2002. J Clin Invest109:1101) and were added to the upper chamber of each well in a totalvolume of 150 gL of DMEM containing 0.5% FCS. Cell populations wereshown to be >99.9% pure by flow cytometric analysis (data not shown).Human SDF-1 (PeproTech Inc., Rocky Hill, N.J., USA) was used atconcentrations of 100 ng/ml, 1 and 10 gg/ml in the lower, upper, or bothlower and upper chambers of the transwell to generate a checkerboardanalysis matrix of positive, negative, and absent gradients of SDF-1,respectively. Thymocyte subpopulations were also pretreated with PTX(100 ng/ml) or anti-CXCR4 monoclonal antibody (10 μg/ml) as previouslydescribed and prior to addition into the transmigration assay(Poznansky, M. C., et al. 2002. J Clin Invest 109:1101).

II. Results

A. The CXCR4 Antagonist AMD3100 Impairs Normal CD4 Cell Emigration fromFetal Thymus Organ Culture (FTOC)

The specific non-peptide CXCR4 antagonist, AMD3100, a bicyclamderivative first described for its potent activity against HIV infection(Hatse, S., et al. 2002. FEBS Left 527:255; De Clercq, E., et al. 1992.Proc Natl Acad Sci USA 89:5286), has been shown to inhibit intracellularCa+ flux and chemotactic responses of murine splenocytes to SDF-1(Matthys, P., et al. 2001. J Immunol 167:4686). CXCR4 expression wasmeasured in SP and DP thymocytes from day 14 FTOC (not shown). Asexpected, CXCR4 was expressed at a higher level in DP thymocytes (MFI52.1±11.3) compared to SP cells. SP CD4 thymocytes showed significantlyhigher expression of CXCR4 compared to SP CD8 cells (MFI 29.1±6.5 vs.15.5±7.3, respectively, p=0.01). FTOC at day 14 of culture wereincubated for 24 hours with 1 μg/ml of AMD3100. Treatment of FTOC with 1μg/ml AMD3100 significantly increased the total number of cells in thethymus as compared to untreated FTOC (FIG. 15A; 349±99.5 vs.247±72.9×10³/lobe, respectively, p=0.022). The total number of SP CD8emigrants did not differ between AMD3100-treated and untreated FTOC(p=0.93). However, in contrast, SP CD4 thymocytes were severely impairedin their ability to emigrate from AMD3100-treated thymuses as comparedto untreated controls (FIGS. 15A, C, 1.1±0.15 vs. 4.07±0.12×10³/lobe,respectively; p=0.005). Analysis of CD62L expression revealed thatretention of CD62L^(high) CD4 SP cells in the thymus of AMD3100-treatedFTOC had occurred in comparison to untreated CXCR4+/+thymuses (FIGS.15B, D; p=0.017). CD62L expression by SP CD8 thymocytes was not affectedin AMD3100 treated thymuses (FIGS. 15B, D; p=0.56). In effect, blockadeof the CXCR4 receptor in normal thymocytes by AMD3100 led to theretention of mature T cells in the thymus, in vitro and in vivo. Thus,the anti-fugetactic agent AMF3100 also works to inhibit fugetaxis of Tcells emigrating from the thymus.

III. Further Description of the Drawings

FIG. 15: CXCR4^(+/−) E15.5 FTOC (n=4) were cultured for 14 days and thentreated for 24 hours with 1 μg/ml AMD3100. Thymic cells and RTE werecollected and stained for CD4 and CD8 (A, C) and CD62L (B, D). Theabsolute number (mean±SEM) of SP CD4 and CD8 RTE (C, *p=0.005) and thefraction (mean±SEM) of CD62L^(high) SP intrathymic thymocytes (D,**p=0.017) from AMD3100-treated and untreated FTOC are shown.

Example III: Protein Kinase C Inhibitors Have Anti-Fugetactic Effects onNeutrophils

Neutrophil chemotaxis can serve as a prototype for understanding highereukaryotic cell migration and gradient sensing (Iijima, M., et al. 2002.Dev Cell 3:469-78; Parent, C. A., et al. 1999. Science 284:765-70). Itis well established that neutrophils undergo chemoattraction, orpersistently directionally biased movement towards a number ofchemokinetic agents elaborated at sites of tissue injury, including thechemokine IL-8 (Baggiolini, M., et al. 1992. FEBS Lett 307:97-101; Rot,A. 1993. Eur J Immunol 23:303-6; Luster, A. D. 1998. N Engl J Med338:436-45). It has now been determined that neutrophil fugetaxis can beabrogated by anti-fugetactic Protein Kinase C inhibitors such asGF109203X.

I. Materials and Methods

A. Neutrophil Isolation

Human whole blood was obtained from healthy volunteers by venipunctureinto tubes containing sodium heparin (Becton Dickinson, San Jose,Calif.). Neutrophils were isolated by density gradient centrifugationand purified by hypotonic lysis, as previously described (Tager, A. M.,et al. 1998. Am J Respir Cell Mol Biol 19:643-52).

B. Fabrication, Preparation, and Characterization of Microfluidic LinearGradient Generator

The microfluidic linear gradient generators were fabricated inpoly(dimethylsiloxane) (PMDS; Sylgard 184, Dow Corning, N.Y.), andgradient generation was verified with fluorescin isothiocyanate (FITC;Sigma-Aldrich), as previously described (Li Jeon, N., et al. 2002. NatBiotechnol 20:826-30; Whitesides, G. M., et al. 2001. Annu Rev BiomedEng 3:335-73).

C. Microfluidic Migration Assay and Time-Lapse Microscopy

Neutrophils at a concentration of 5×10⁶ cells/ml were loaded uniformlyacross the migration channel and allowed to migrate in the absence orpresence of a linear gradient of human Interleukin-8 (72 amino acids;Pepro-Tech, Rocky Hill, N.J.) in Iscove's Modified Dulbecco's Medium(IMDM; Mediatech, Herndon, Va.) with 0.5% (w/v) fetal bovine serum (FBS;Mediatech) flowing at 0.1 mm/sec, with peak concentrations of 12 nM, 120nM, and 1.2 μM. Migration was observed in a Nikon Eclipse TE2000-Smicroscope (Nikon, Japan). Brightfield images (10×) were taken every 30seconds using a digital camera (Hamamatsu, Japan) controlled by IPLab3.6.1 (Scanalytics, Fairfax, Va.). Cell movement and gradientverification were always observed at a set point along the migrationchannel. Secondary effects of chemokinetic agents secreted by cellsduring the course of the experiments were effectively ruled outpreviously and were considered unlikely in view of the flow rate offluid through the device throughout the assay. In certain experiments,cells were pre-incubated with GF109203X (25 mM for 30 minutes at 25°C.), and then washed and loaded into the device.

D. Mathematical Analysis of Cell Migration in Linear Gradient Generator

Cell movement was tracked using MetaMorph 4.5 (Universal Imaging,Dowington, Pa.) object tracking application, which generated tables ofCartesian coordinate data for each cell. Tracking data was analyzed inExcel (Microsoft, USA) and MATLAB 13 (The Mathworks, Inc., Nowton,Mass.) to determine angular frequencies, mean squared displacements,migration velocities, persistence times, random motility coefficients,and random walk path lengths. Cell movement was characterized based on apersistent random walk model as follows (Moghe, P. V., et al. 1995. JImmunol Methods 180:193-211; McCutcheon, M. 1946. Physiological Reviews26:319). For each cell, the squared displacement R(t) was calculated attime interval t,R ²(t)=(x(t ₀ +t)−x(t ₀))²+(y(t ₀ +t)−y(t ₀))²,  (1)where t₀ is the time at the origin. The origin was shifted along thedata set and the displacements were averaged for overlapping timeintervals. A global average was performed over all cells in the set tocalculate the mean squared displacement. Mathematically modeling cellmovement as a correlated, biased random walk, this can be written asR ²(t)=2S ² P[t−P(1−e ^(−t/P))],  (2)where S and P are measures of the mean speed of movement and persistencetime respectively. When time interval t is much greater than persistencetime P, the mean squared displacement becomes linearly proportional to1, analogous to Brownian diffusion,R ²(t)=2S ² Pt=4μt3,  (3)where μ is the motility coefficient. The slope and intercept of a leastsquares regression fitted to the linear section of the mean squareddisplacement give an estimate of μ and P, respectively. Further, a“persistence index” (PI) of the motion or mean free path, was calculatedas the total displacement of the cell divided by the total path length.The PI is an indicator of turning behavior, with 1 indicating motion ina straight line and 0 indicating no net displacement. To quantifydirectional bias of neutrophil migration in defined IL-8 gradients inmicrofabricated devices, we calculated a “chemotropism index” (CI) basedon McCutcheon, et al. (Mc Cutcheon, M. 1946. Physiological Reviews 26,319; Nossal, R., et al. 1976. Biophys J 16, 1171-82) and defined as thenet path length traversed by a cell with respect to the direction of theestablished gradient divided by the total distance traveled and modifiedto include a measurement of directionality towards or away from themaximal chemokine concentration:

$\begin{matrix}{{CI} = \frac{\sum{l_{i}\cos\;\varphi_{i}}}{\sum l_{i}}} & (4)\end{matrix}$where, li is the length of a cell's movement vector and φ_(i) is theangle the movement vector makes with respect to the establishedgradient. The CI is therefore an indicator of accuracy of orientationwith respect to the gradient and will be 1 if a cell moved directly upthe gradient, 0 if there is no preferred orientation, and −1 formigration directly down the gradient. The index was calculated for eachcell and then averaged over the population of migrating cells to obtainthe “mean chemotropism index” (MCI).

II. Results

A. Protein Kinase C Inhibitor GF109230X Impacts Neutrophil Migration

In an effort to determine if Protein Kinase C (PKC) secondary messengersignal amplification or dampening could control directional bias ofneutrophil fugetaxis, and in light of the finding that calcium-dependentPKCs have been shown to be involved in directional cell migration (Jin,M. et al. 2005. J Neurosci 25:2338-47; Sun, X. G., et al. 1999. CellGrowth Differ 10:343-52; Battle, E., et al. 1998. J Biol Chem273:15091-8; Carnevale, K. A., et al. 2003. J Biol Chem 278:25317-22),neutrophils were pretreated with the selective calcium-dependent PKCinhibitor, GF109203X (Toullec, D., et al. 1991. J Biol Chem266:15771-81), and then exposed to gradients of IL-8 in which neutrophilfugetaxis (600 nM to 1.2 mM) or chemoattraction was predominant (0 to120 nM). GF109230X at a concentration of 25 μM selectively inhibitedfugetaxis and converted a fugetactic response into a predominantlychemoattractant response (FIG. 16) (p<0.0001). Thus, PKC inhibitors alsofunction as anti-fugetactic agents.

III. Further Description of the Drawings

FIG. 16: The mean chemotropism index (MCI) (+/− standard error) for eachindicated condition are shown for each IL-8 gradient condition includingin the absence or presence of an IL-8 gradient in the presence of GF109230X, 8-Br-cAMP, 8-Br-cGMP, Rp-cAMPS, and Rp-8-Br-cGMPS. The numberof cell tracks analysed to generate the MCI are shown in brackets to theright of the figure and are derived from triplicate experiments for eachcondition. MCI values less than −0.1 indicate directionally biased downthe gradient (fugetaxis) and MCI values greater than +0.1 indicatedirectionally biased movement up the gradient (chemotaxis orattraction). †p<0.005 or *p<<0.0001: Student t text comparison of MCIfor each inhibitor condition as compared to the relevant controlgradient without inhibitor.

Example IV: Assay for Fugetactic and Anti-Fugetactic Agents

T-cells are repelled by fugetactic agents in a concentration-dependentmanner. In culture, repulsion of T-cells by fugetactic agents results inthe formation of a zone of clearance around the cells. When pre-treatedwith an anti-fugetactic inhibitor, the zone of clearance does not formaround T-cells despite the presence of the fugetactic agent.Accordingly, a binary assay for fugetactic and anti-fugetactic agents isdescribed herein.

I. Materials and Methods

A. Formation of SDF-1 Gradients

For the analysis of T-cell responses in SDF-1 gradients generated byβ-TC3 cells, murine T-cells were added to microwells with SDF-1secreting and control β-TC3 cells, preliminary grown as round 6 mmpatches on fibronectin or laminin coated 24-well and 48-well plates (BDBioSciences, Bedford, Mass.), incubated for 15 hours at 37° C. andobserved using timelapse videomicroscopy. Images were taken every 30seconds using a Hamamatsu camera (Hamamatsu, Japan) controlled by IPLabsoftware. The CXCR4 receptor was blocked by pre-incubation of T-cellswith 5 μg/ml AMD3100 (Sigma, St. Louis, Mo.) for 30 min at 37° C. aspreviously described (70). Trajectories of migration were determined forrandomly selected T-cells using Metamorph software. Cell path trackingand mean chemotropic indices (MCI) as a measurement of directionality ofmovement towards or away from β-TC3 cells were calculated usingMetaMorph and MatLab software as previously described where an MCI of+0.1 to −0.1 indicates chemokinesis, >+0.1 indicates chemotaxis and<−0.1 indicates fugetaxis (Moghe, P. V., et al. 1995 J Immunol Methods180, 193-211; Mc Cutcheon, M. (1946) Physiological Reviews 26, 319).

II. Results

An assay allowing examination of the fugetactic effect of a steepcontinuous SDS-1 gradient on T-cell migration has been designed, whereT-cells were added to MSCV.SDF-1 bright cells or control cells and theiractive movement observed directly for 15 hours using time-lapsemicroscopy. T-cells were evenly distributed within the wells withcontrol MSCV cells as well as SDF-1 secreting MSCV.SDF-1 bright cells atthe beginning of experiments. In the wells with control cells,distribution of T-cells remained random by the end of the experiment andcell migration was documented to be chemokinetic in nature by time-lapsevideo microscopy and by cell path tracking and MCI (−0.018+/−0.012)(FIGS. 17A and D). In contrast, T-cells migrated away from MSCV.SDF-1bright cells and formed zones of clearance of T-cells around the patchesof SDF-1 secreting cells at 15 hours of incubation. T-cells tended tomigrate towards SDF-1 secreting cells during the first 1.5 hours ofincubation (MCI=+0.08+/−0.018) but subsequently changed to a fugetacticresponse (MCI=−0.268+/−0.019) (FIGS. 17B and E). To confirm thatfugetaxis of T-cells was directly related to SDF-1 secretion byMSCV.SDF-1 bright cells, T-cells were pretreated with the specific CXCR4antagonist AMD3100. T-cells treated in this way failed to migrate awayfrom SDF-1 secreting cells and remained randomly distributed aroundMSCV.SDF-1 bright cells (FIG. 17C). In this way, it has been shown thatMSCV.SDF-1 bright cells and not control. MSCV cells elicited migrationalresponses from T-cells, and that the high level of SDF-1 secretion byMSCV.SDF-1 bright cells was sufficient to repel T-cells in vitro via aCXCR4 mediated mechanism.

III. Further Description of the Drawings

FIG. 17: Demonstration of T-cell repulsion from MSCV.SDF-1 bright cellsproducing high levels of SDF-1 using time-lapse microscopy. Pan-T cellswere randomly distributed in the wells with control MSCV or MSCV.SDF-1bright cells and incubated for 15 hours. FIGS. 24a-24c represent finaldistribution of T-cells by the end of experiment, which remained randomin the wells with control MSCV cells (A) and MSCV.SDF-1 bright cellswhen T-cells were pretreated with CXCR4 antagonist AMD3100 (C), whereasthe repulsion of untreated T-cells from MSCV.SDF-1 bright cellsproducing high levels of SDF-1 cells resulted in formation of a“clearance zone” (B). FIG. 17D-E represent migratory paths of untreatedT-cells observed within first 2.5 hours of co-incubation with controlMSCV (D) or MSCV.SDF-1 bright cells (E). The start points of migrationtracks are distributed along the axis, the end points are marked withcrosses. In the control MSCV wells T-cell migration was chemokinetic bynature whereas in the wells with MSCV.SDF-1 bright cells T-cellsmigration was chemotactic towards SDF-1 secreting cells within firsthour but subsequently changed to fugetactic.

Example V: Fugetactic Effects of Ligand Dimerization

High levels of intracytoplasmic calcium flux in neutrophils are shownherein to be associated with the fugetactic action of chemokines.Drawing parallels between these conditions of intracytoplasmic calciumflux and the conditions under which the fugetactic agents IL8 and SDF-1are dimerized indicates that such agents are dimerized when mediatingtheir fugetactic effects.

I. Materials and Methods

-   -   A. Materials and methods for the isolation of neutrophils are        described in Part I of Example III, above.    -   B. Intracellular Calcium Measurements and CXCR2 staining        following IL-8 105

Calcium flux in neutrophils in response to IL-8 was measured aspreviously described (Mahon, M. J., et al. 2004 J Biol Chem279:23550-8). Briefly, isolated cells (˜10⁷/ml) were transferred into abalanced salt solution (127 mM NaCl, 3.8 mM KCl, 1.2 mM KH₂PO₄, 1.2 mMCaCl₂, 0.8 mM MgCl₂, 5 mM glucose, and 10 mM HEPES, pH 7.4), incubatedfor 45 min at room temperature with 1.25 μM Fura-2 acetoxymethyl ester(Molecular Probes, Eugene, Oreg.), washed with the balanced saltsolution, and unloaded for 15 min at room temperature. Cells were thenwashed twice with the balanced salt solution, resuspended at 10⁶/mL, andplated in Lab-Tek II chambered cover glass (Nalge Nunc International,Naperville, Ill.). Calcium flux in response to indicated concentrationsof IL-8 (Akahoshi, T., et al. 1994 Lymphokine Cytokine Res 13:113-6) wasmeasured by fluorescence of cells excited at 340 and 380 nm using a PTIDeltascan dual-wavelength fluorimeter (Photon Technologies Incorporated,Lawrenceville, N.J.), observed through a Nikon Diaphot 200 microscope(Melville, N.Y.) and Sensys charge-coupled device camera (Photometrics,Ltd., Tucson, Ariz.), and analyzed using the Poenie-Tsien ratio withImagemaster 2 software (Photon Technologies Incorporated). Signalingresponses to IL-8 at designated concentrations were calibrated againstmaximal calcium flux as determined by treatment with 5 μM ionomycin(Sigma). CXCR2 expression on neutrophils pre- and post-exposure to 120nM, 1.2 μM and 2.4 μM IL-8 was determined using PE-labelled anti-CXCR2IgG1 antibody (R & D systems). Neutrophils were also stained with aPE-labelled IgG1 isotype control antibody to quantify non-specificstaining. Mean fluorescent intensity (MFI) was determined using FloJosoftware (Tree Star, San Carlos, Calif.). Statistical differencesbetween MFI values generated from isotype control staining and specificCXCR2 staining under experimental conditions was determined byKolmogrov-Smirnoff test.

II. Results

A. Low and High Concentrations of IL-8 Generate Differential Levels ofCalcium Flux

Measurement of intracellular calcium flux serves as an indicator of themagnitude of signal transduction by a GPCR, and both calcium and cyclicnucleotides can serve as secondary messengers of these signals. In orderto ascertain whether signaling output could be generated by chemotacticand fugetactic IL-8 doses, cells were loaded with 1.25 μM Fura2-AM,exposed to 120 nM, 1.2 μM, or 2.4 μM IL-8, and ratio of calcium-boundFura2-AM to unbound was measured by fluorimeter in real time. Peakcalcium flux responses were almost two-fold greater for 1.20 μM(83%+/−7.9%: percentage of maximal calcium flux) than for 120 nM IL-8(48%+/−5.1%) (p<0.05). Previous reports had revealed maximal calcium 60fluxes up to 100 nM concentrations without exploring higherconcentrations. The maximal peak of calcium flux was seen at chemokineconcentrations of 1.2 μM and 2.4 μM and was associated with differentialrates of recovery, supporting the view that the cell could discriminatebetween these concentrations of the chemokine (FIG. 18). Interestingly,the recovery periods for all doses of IL-8 tested demonstrateddifferential levels of bound Fura2-AM, indicating different levels ofintracellular calcium flux after each peak period. Thus, signalmagnitude during the recovery period, as measured by calcium fluxthrough CXCR2 and CXCR1, was greater with increasing concentrations ofIL-8, indicating that neutrophils are capable of detecting andgenerating differential intracellular responses to concentrations ofIL-8 above 100 nM. Differential levels of calcium flux to concentrationsof a chemokine that are known to induce directional migratory responsesand at least 10-fold greater than the estimated Kd of the recombinantchemokine receptor have been clearly demonstrated for the chemokine,SDF-1 (CXCL12) and its interaction with CXCR4 (Princen, K., et al. 2003Cytometry A 51:35-45).

In order to determine whether signal output as measured by calcium fluxcould be generated from sequential low or high doses of IL-8,neutrophils were exposed to two pulses of IL-8 at low and highconcentration (FIGS. 18A-D). Sequential calcium fluxes were seen incells exposed to repeated pulses of low dose (120 nM) which had beenseen to induce maximal chemoattraction (FIG. 18A) and high dose (1.2 μM)IL-8 (FIG. 18B) and a dose of 600 nM followed by a dose of 1.24 μM (FIG.18C) which were associated with a maximal fugetactic response.Sequential calcium flux was not seen when cells were exposed to thehighest dose of 2.4 μM, and this was then followed by a further highconcentration of chemokine, implying that receptor saturation occurredat concentrations above 1.2 μM (FIG. 18D). These data were consistentwith the finding that neutrophils were capable of directional migrationand gradient sensing up to a peak concentration of 1.2 μM but underwentonly chemokinetic movement in gradients with peak concentrations abovethis.

Given the presence of a sequential calcium flux with fugetacticconcentrations of IL-8, it was examined whether CXCR2 was stilldetectable on the surface of the neutrophil after the first pulse ofchemokine. Neutrophils were immunostained for CXCR2 following a singlepulse of IL-8 at concentrations of 12 nM, 120 nM, 1.2 μM and 2.4 μM.CXCR2 was downregulated from baseline expression levels immediatelyafter chemokine exposure, with both chemoattractant and fugetacticconcentrations of IL-8 with consistent 2- to 3-fold reductions in meanfluorescent intensities (data not shown) and remained 4- to 5-foldgreater than MFI values for control isotype antibody staining (p<0.01)(data not shown). CXCR2 was clearly still detectable on the cell surfacefollowing exposure to low or high concentrations of IL-8.

In view of the effect of the ability of picomolar concentrations of thenon=peptide CXCR2 antagonist SB225002 (Calbiochem, CA) to influenceneutrophil directional decision-making, it was examined whether ultralowconcentrations of SB225002 could reduce the level of sequential calciumflux to high concentrations of chemokine. Treatment with 1 pM SB225002significantly reduced sequential peaks of calcium flux followingstimulation with concurrent doses of IL-8 at concentrations of 1.2 μMand 1.2 μM (FIG. 18B) (p<0.05) or 600 nM and 1.2 μM (FIG. 18C) (p<0.05),as compared to calcium flux magnitudes in the absence of the receptorantagonist. The magnitude of calcium flux following SB225002 treatmentin conditions shown in (FIG. 18B) and (FIG. 18C) were of similar size tothose seen with sequential pulses of lower concentrations of chemokineassociated with a chemoattractant response. These data validate theobserved exquisite sensitivity of receptor-mediated generation ofdirectional bias in the motile response of cells exposed to fugetacticconditions.

Chemokines, including IL-8 and SDF-1 can serve as fugetactic agents atconcentrations above 100 nM. Moreover, at such concentrations IL-8 andSDF-1 are known to exist in a dimerized state. (Lowman, H. B. et al.,1997 Protein Science 6:598-608; Veldkamp, C. T. et al. 2005 ProteinScience 14:1071-1081; Horcher, M. et al. 1998 Cytokine 10:1-12). Asshown herein, high levels of chemokine also induce intracytoplasmiccalcium flux in neutrophils, which is associated with the fugetacticaction. Thus, a signaling paradigm can be envisioned, wherein binding ofa dimeric chemokine and dimerization of the cognate receptor on the cellsurface elicits a differential signal in the cell, including an increasein calcium flux, which result in the fugetactic response.

III. Further Description of the Drawings

FIG. 18: Differential Calcium Flux. Primary human neutrophils wereloaded with 1.25 μM Fura2-AM and exposed to sequential pulses ofchemokine 120 seconds apart (A-C) and calcium flux measured byfluorimeter. Calcium flux in neutrophils was monitored followingexposure to a single pulse of chemokine at a concentration of 120 nMfollowed by a subsequent pulse of 120 nM (A), 1.2 μM followed by 1.2 μM(B), 600 nM followed by 1.2 μM (C) or 2.4 μM followed by 2.4 μM (D)(solid lines). Calcium flux was also monitored in neutrophils pretreatedwith 1 pM SB225002 prior to treatment, as in (B) and (C) (dotted lines).The results of a representative experiment of three that were performedin this way are shown. Initial and secondary pulses of chemokine(indicated by arrows along the y-axis) are shown.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications can be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention, which is delineated by the appended numbered claims.

Each of the applications and patents cited in this text, as well as eachdocument or reference cited in each of the applications and patents(including during the prosecution of each issued patent; “applicationcited documents”), and each of the PCT and foreign applications orpatents corresponding to and/or claiming priority from any of theseapplications and patents, and each of the documents cited or referencedin each of the application cited documents, are hereby expresslyincorporated herein by reference. More generally, documents orreferences are cited in this text, either in a Reference List before theclaims, or in the text itself; and, each of these documents orreferences (“herein-cited references”), as well as each document orreference cited in each of the herein-cited references (including anymanufacturer's specifications, instructions, etc.), is hereby expresslyincorporated herein by reference.

REFERENCES

-   1. Chaffin K E and Perlmutter R M. A pertussis toxin-sensitive    process controls thymocyte emigration. Eur. J. Immunol. 21:    2565-2573 (1991)-   2. Craddock C F, Nakamoto B, Andrews R G, Priestley G V and    Papayannopoulou T Antibodies to VLA4 integrin mobilize long-term    repopulating cells and augment cytokine-induced mobilization in    primates and mice. Blood 90, 4779-4788 (1997)-   3. Doetsch R N and Seymour W F. Negative chemotaxis in bacteria.    Life Sciences 9:1029-1037 (1970)-   4. Bailey G B, Leitch G J and Day D B. Chemotaxis by entamoeba    histolytica. J Protozool 32:341-346 (1985)-   5. Tsang N, Mcnab R and Koshland D E Jr. Common mechanism for    repellents and attractants in bacterial chemotaxis. Science    181:60-69 (1973)-   6. Repaske D and Adler J. Change in intracellular pH of Escherischia    coli mediates the chemotactic response to certain attractants and    repellents. J Bacteriol 145:1196-1208 (1981)-   7. Tisa L S and Adler J. Cytoplasmic free-Ca2+ level rises with    repellents and falls with attractants in Escherischia coli    chemotaxis. Proc Natl Aca Sci U.S.A. 92:10777-10781 (1995)-   8. Taylor B L and Johnson M S. Rewiring a receptor: negative output    from positive input. FEBS Lett 425:377-381 (1998)-   9. Wells T N. Power C A and Proudfoot A E. Definition, function and    pathophysiological significance of chemokine receptors. Trends    Pharmacol Sci 19:376-380 (1998)-   10. Luster A D. Chemokines—chemotactic cytokines that mediate    inflammation. N Engl J Med 338:436-445 (1998)-   11. Baggiolini M. Chemokines and leukocyte traffic. Nature    392:565-568 (1998)-   12. Bleul C C, Fuhlbrigge R C, Casasnovas J M, Aiuiti A and Springer    T A. A highly efficacious lymphocyte chemoattractant, stromal    cell-derived factor 1 (SDF-1). J Exp Med 184: 1101-1109 (1996)-   13. Kim C H, Pelus L M, White J R and Broxmeyer H E. Differential    chemotactic behavior of developing T-cells in response to thymic    chemokines. Blood. 91: 4434-4443 (1998)-   14. Tashiro K, Tada H, Heilker R, Shirozu M, Nakano T and Honjo F.    Signal sequence trap: cloning strategy for secreted proteins and    type 1 membrane proteins. Science, 261: 600-603-   15. Shirozu M, Nakano T, Inazawa J, Tashiro K, Tado H, Shinohara T    and Honjo T. Structure and chromosomal localization of the human    stromal cell-derived factor 1 (SDF1) gene. Genomics 28:495-500    (1995)-   16. Bacon K B, Premack B A, Gardner P and Schall T J. Activation of    dual T cell signaling pathways by the chemokine RANTES. Science,    269:1727-30 (1995)-   17. Noble P B and Bentley K C. Locomotory characteristics of human    lymphocytes undergoing negative chemotaxis to oral carcinomas. Exp    Cell Res 133; 457-461 (1981)

We claim:
 1. A method for increasing the migratory capacity of immune cells into a mammalian ovarian tumor exhibiting fugetactic activity on said immune cells due to CXCL12 secretion, wherein said immune cells comprise tumor antigen-specific T-cells, which method comprises: a) selecting a mammal having said tumor which secretes a fugetactic effective amount of CXCL12 around said tumor wherein said concentration of CXCL12 is greater than 100 nM; b) contacting said T-cells with an anti-fugetactic concentration of 1,1′-[1,4-phenylenebis-(methylene)]bis[1,4,8,11-tetraazacyclotetradecane] of from 0.08 μg/mL up to 5 μg/mL wherein said T-cells are now capable of increasing their migration into said tumor through the fugetactic activity of said tumor.
 2. The method of claim 1, wherein said method further comprises administering anti-cancer therapy to the said patient.
 3. The method of claim 2, wherein said anti-cancer therapy is a chemotherapeutic agent selected from the group consisting of paclitaxel, cisplatin, and doxorubicin.
 4. The method of claim 3, wherein the chemotherapeutic agent is paclitaxel.
 5. The method of claim 1, wherein the anti-fugetactic agent is in a sustained release formulation.
 6. The method of claim 1, wherein the mammal is a human. 